Methods of increasing root biomass in plants

ABSTRACT

The invention provides methods and materials for increasing root biomass in a plant, by increasing the expression of at least one PEAPOD protein, or fragment thereof, in the plant. The invention also provides methods and materials for producing a plant increased root biomass, the method comprising the step of increasing the expression of at least one PEAPOD protein, or fragment thereof, in the plant.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage Application filed under 35 U.S.C. § 371 of International Application No. PCT/IB2015/058479, filed Nov. 3, 2015, which claims the benefit of New Zealand Application No. 701643, filed Nov. 4, 2014. Both of these applications are hereby incorporated by reference in their entireties.

TECHNICAL FIELD

The present invention relates methods for producing plants with increased root biomass.

BACKGROUND ART

The roots of vascular plants have multiple functions. These include: 1) absorption of water and inorganic nutrients, 2) anchoring of the plant to the ground, and supporting it, 3) storage of food and nutrients, and 4) vegetative reproduction. In response to the concentration of nutrients, roots also synthesise cytokinin, which acts as a signal influencing how fast the shoots can grow.

Plants with increased root biomass would therefore potentially have a number of advantages including better anchorage, more efficient water uptake, more efficient nutrient uptake, and improved drought tolerance. A combination of these features may also result in improved yield, including increased grain or fruit biomass and/or increased leaf biomass.

At present there is limited understanding of the genetic mechanisms controlling root biomass in plants.

It would be beneficial to have available alternative methods for controlling root biomass in plants.

It is therefore an object of the invention to provide methods and materials for increasing the production of root biomass in plants, and/or at least to provide the public with a useful choice.

SUMMARY OF THE INVENTION

Previously, White (2006) discovered two adjacent homologous genes in Arabidopsis (named PEAPOD, PPD1 and PPD2) that regulate the cell proliferation of meristemoids during the late stages of leaf and seed pod development. Deletion of these genes in Arabidopsis resulted in enlarged leaves and wide seed pods while over expression of PPD1 resulted in a reduction in the size of the leaves and siliques (White, 2006).

The applicants have now surprisingly shown that the over-expression of PEAPOD genes in plants results in an increase in the production of root biomass.

The applicant's invention therefore relates to a method for increasing root biomass in plants by ectopic expression of PEAPOD. In particular the invention relates to ectopic expression of PEAPOD proteins that are characterized by presence of consensus amino acid motifs common to all PEAPOD proteins disclosed from a wide range of plant species.

Methods

In the first aspect the invention provides a method for increasing root biomass in a plant, the method comprising the step of increasing the expression of at least one PEAPOD protein in the plant.

In one embodiment root biomass is increased relative to that in a control plant, of the same species or variety.

In one embodiment the increased expression of the at least one PEAPOD protein is a consequence of the plant, or its ancestor plant or plant cell, having been transformed with a polynucleotide encoding the PEAPOD protein.

In a further embodiment, the plant is transgenic for at least one polynucleotide encoding and expressing the PEAPOD protein.

In a further aspect the invention provides a method for producing a plant increased root biomass, the method comprising the step of increasing the expression of at least one PEAPOD protein in the plant.

In one embodiment the plant is transformed with at least one polynucleotide encoding a PEAPOD protein.

In a further embodiment the method comprises the step of transforming the plant, or transforming a plant cell which is regenerated into the plant, with a polynucleotide encoding the PEAPOD protein.

In one embodiment the method includes the additional step of testing or assessing the plant for increased root biomass.

In a further embodiment the method includes the step producing further plants with increased root biomass by asexually or sexually multiplying the plants tested for increased biomass.

PEAPOD Proteins

In one embodiment the PEAPOD protein is a polypeptide comprising the sequence of at least one of SEQ ID NO: 28, 29, 31, 32, 34 and 35.

In a further embodiment the PEAPOD protein comprises the sequence of SEQ ID NO: 28. In a further embodiment the PEAPOD protein comprises the sequence of SEQ ID NO: 29. In a further embodiment the PEAPOD protein comprises the sequence of SEQ ID NO:31. In a further embodiment the PEAPOD protein comprises the sequence of SEQ ID NO:32. In a further embodiment the PEAPOD protein comprises the sequence of SEQ ID NO:34. In a further embodiment the PEAPOD protein comprises the sequence of SEQ ID NO:35.

In a further embodiment the PEAPOD protein is a polypeptide comprising a sequence with at least 70% identity to any one of SEQ ID NO: 1 to 26.

In a further embodiment the PEAPOD protein is a polypeptide comprising a sequence selected from any one of SEQ ID NO: 1 to 26.

In a further embodiment the PEAPOD protein is a polypeptide comprising a sequence with at least 70% identity to SEQ ID NO: 1.

In a further embodiment the PEAPOD protein is a polypeptide comprising the sequence of SEQ ID NO: 1.

Expressing PEAPOD

Methods for expressing proteins in plants are well known to those skilled in the art, and are described herein. All of such methods are included within the scope of the invention.

Increasing Expression of PEAPOD by Introducing a Polynucleotide

In one embodiment expression is increased by introducing at least one polynucleotide into the plant cell or plant.

In a preferred embodiment the polynucleotide encodes a PEAPOD protein as herein defined.

In a further embodiment the polynucleotide comprises a sequence with at least 70% identity to the coding sequence of any one of SEQ ID NO: 83-107.

In a further embodiment the polynucleotide comprises a sequence with at least 70% identity to the sequence of any one of SEQ ID NO: 83-107.

In a further embodiment the polynucleotide comprises the coding sequence of any one of SEQ ID NO: 83-107.

In a further embodiment the polynucleotide comprises the sequence of any one of SEQ ID NO: 83-107.

In a further embodiment the polynucleotide comprises a fragment of the sequences described above, that is capable of encoding a polypeptide with the same function as a PEAPOD protein. In one embodiment the fragment encodes a polypeptide capable of increasing root biomass.

Expressing PEAPOD via an Expression Construct

In a preferred embodiment the polynucleotide is introduced into the plant as part of an expression construct.

In a preferred embodiment the expression construct comprises a promoter operatively linked to the polynucleotide.

Promoter for Increasing Expression of PEAPOD

In one embodiment the promoter is capable of driving, or drives, expression of the operatively linked polynucleotide constitutively in all tissues of the plant.

In a further embodiment the promoter is a tissue-preferred promoter.

In a further embodiment the promoter is capable of driving, or drives, expression of the operatively linked polynucleotide in the below ground tissues of the plant.

In one embodiment the promoter is a below ground tissues-preferred promoter.

In a further embodiment the promoter is a below ground tissue-specific promoter.

In one embodiment the promoter is a light-repressed promoter.

In a further embodiment the promoter is capable of driving, or drives, expression of the operatively linked polynucleotide in the roots of the plant.

In one embodiment the promoter is a root-preferred promoter.

In a further embodiment the promoter is a root-specific promoter.

Source of Polynucleotides and Polypeptides

The polynucleotides and variants of polynucleotides of the invention, or used in the methods of the invention, may be derived from any species. The polynucleotides and variants may also be synthetically or recombinantly produced, and also may be the products of “gene shuffling” approaches.

The polypeptides and variants of polypeptides of the invention, or used in the methods of the invention, may be derived from any species. The polypeptides and variants may also be recombinantly produced and also may also be expressed from the products of “gene shuffling” approaches.

In one embodiment the polynucleotide, polypeptide or variant, is derived from a plant species.

In a further embodiment the polynucleotide, polypeptide or variant, is derived from gymnosperm plant species.

In a further embodiment the polynucleotide, polypeptide or variant, is derived from an angiosperm plant species.

In a further embodiment the polynucleotide, polypeptide or variant, is derived from a dicotyledonous species.

In a preferred embodiment the polynucleotide, polypeptide or variant, is derived from a eudicot species.

In a further embodiment the polynucleotide, polypeptide or variant, is derived from a eudicot plant species.

In a further embodiment the polynucleotide, polypeptide or variant, is derived from a monocotyledonous species. Preferred monocot plants include: palm, banana, duckweed and orchid species.

Plant Cells and Plants to be Transformed

The plant cells and plants of the invention, or used in the methods of the invention, are from any plant species.

In one embodiment the plant cells or plants are from gymnosperm plant species.

In a further embodiment the plant cells or plants are from angiosperm plant species.

In a further embodiment the plant cells or plants are from a dicotyledonous species.

Preferred monocotyledonous genera include: Agropyron, Allium, Alopecurus, Andropogon, Arrhenatherum, Asparagus, Avena, Bambusa, Bothrichloa, Bouteloua, Bromus, Calamovilfa, Cenchrus, Chloris, Cymbopogon, Cynodon, Dactylis, Dichanthium, Digitaria, Eleusine, Eragrostis, Fagopyrum, Festuca, Helianthus, Hordeum, Lolium, Miscanthis, Miscanthus×giganteus, Oryza, Panicum, Paspalum, Pennisetum, Phalaris, Phleum, Poa, Saccharum, Secale, Setaria, Sorgahastum, Sorghum, Triticum, Vanilla, X Triticosecale Triticale and Zea.

Preferred monocotyledonous species include: Agropyron cristatum, Agropyron desertorum, Agropyron elongatum, Agropyron intermedium, Agropyron smithii, Agropyron spicaturn, Agropyron trachycaulum, Agropyron trichophorum, Allium ascalonicum, Allium cepa, Allium chinense, Allium porrum, Allium schoenoprasum, Allium fistulosum, Allium sativum, Alopecurus pratensis, Andropogon gerardi, Andropogon Gerardii, Andropogon scoparious, Arrhenatherum elatius, Asparagus officinalis, Avena nuda, Avena sativa, Bambusa vulgaris, Bothrichloa barbinodis, Bothrichloa ischaemurn, Bothrichloa saccharoides, Bouteloua curipendula, Bouteloua eriopoda, Bouteloua gracilis, Bromus erectus, Bromus inermis, Bromus riparius, Calamovilfa longifilia, Cenchrus ciliaris, Chloris gayana, Cymbopogon nardus, Cynodon dactylon, Dactylis glomerata, Dichanthium annulatum, Dichanthium aristatum, Dichanthium sericeum, Digitaria decumbens, Digitaria smutsii, Dioscorea rotundata, Dicsorea alata, Dicscorea opposita, Dicscorea bulbifera, Dioscorea esculenta, Dioscorea trifida, Eleusine coracan, Elymus angustus, Elymus junceus, Eragrostis curvula, Eragrostis tef, Fagopyrum esculenturn, Fagopyrum tataricum, Festuca arundinacea, Festuca ovina, Festuca pratensis, Festuca rubra, Helianthus annuus sunflower, Hordeum distichum, Hordeum vulgare, Lolium multiflorum, Lolium perenn, Miscanthis sinensis, Miscanthus×giganteus, Oryza sativa, Panicum italicium, Panicum maximum, Panicum miliaceum, Panicum purpurascens, Panicum virgatum, Panicum virgatum, Paspalum dllatatum, Paspalum notatum, Pennisetum clandestinurn, Pennisetum glaucum, Pennisetum purpureum, Pennisetum spicaturn, Phalaris arundinacea, Phleum bertolinii, Phleum pratense, Poa fendleriana, Poa pratensis, Poa. nemoralis, Saccharum officinarum, Saccharum robustum, Saccharum sinense, Saccharum spontaneum, Secale cereale, Setaria sphacelate, Sorgahastum nutans, Sorghastrum nutans, Sorghum dochna, Sorghum halepense, Sorghum sudanense, Sorghum vulgare, Sorghum vulgare, Triticum aestivum, Triticum dicoccum, Triticum durum, Triticum monococcum, Vanilla fragrans, X Triticosecale and Zea mays.

A preferred family of monocotyledonous plants is poaceae family.

Preferred poaceae subfamilies include the: Anomochlooideae, Pharoideae, Puelioideae, Bambusoideae, Pooideae, Ehrhartoideae, Aristidoideae, Arundinoideae, Chloridoideae, Panicoideae, Danthonioideae, and Micrairoideae.

A preferred poaceae family is the subfamily pooideae. Preferred pooideae plants include wheat, barley, oats, brome grass and reed grass.

Another preferred poaceae family is the subfamily Ehrhartoideae. Preferred ehrhartoideae plants include rice.

Another preferred poaceae family is the subfamily panicoideae. Preferred panicoideae plants include panic grass, maize, sorghum, sugar cane, energy cane, millet, fonio and bluestem grasses.

Another preferred poaceae family is the subfamily Arundinoideae. Preferred Arundinoideae plants include Arundo donax.

Another preferred poaceae family is the subfamily Bambusoideae. Preferred Bambusoideae plants include bamboo.

Preferred poaceae species include those form the Lolium genera. Preferred Lolium species include Lolium longiflorum, Lolium multiflorum, Lolium perenne, Lolium westerwoldicum, Lolium temulentum, and Lolium hybridum.

Other preferred poaceae species include those form the Festuca genera. Preferred Festuca species include Festuca arundinacea, Festuca ovina, Festuca pratensis and Festuca rubra.

Preferably the plant cells or plants are from a dicotyledonous species.

Preferred dicotyledonous genera include: Amygdalus, Anacardium, Arachis, Brassica, Cajanus, Cannabis, Carthamus, Carya, Ceiba, Cicer, Cocos, Coriandrum, Coronilla, Cossypium, Crotalaria, Dolichos, Elaeis, lycine, Gossypium, Helianthus, Lathyrus, Lens, Lespedeza, Linum, Lotus, Lupinus, Macadamia, Medicago, Melilotus, Mucuna, Olea, Onobrychis, Ornithopus, Papaver, Phaseolus, Phoenix, Pistacia, Pisum, Prunus, Pueraria, Ribes, Ricinus, Sesamum, Theobroma, Trifolium, Trigonella, Vicia and Vigna.

Preferred dicotyledonous species include: Amygdalus communis, Anacardium occidentale, Arachis hypogaea, Arachis hypogea, Brassica napus Rape, Brassica. nigra. Brassica campestris, Cajanus cajan, Cajanus indicus, Camelina sativa, Cannabis sativa, Carthamus tinctorius, Carya illinoinensis, Ceiba pentandra, Cicer arietinum, Cocos nucifera, Coriandrum sativum, Coronilla varia, Cossypium hirsutum, Crotalaria juncea, Dolichos lablab, Elaeis guineensis, Gossypium arboreum, Gossypium nanking, Gossypium barbadense, Gossypium herbaceum, Gossypium hirsutum, Glycine max, Glycine ussuriensis, Glycine gracilis, Helianthus annus, Jatropha cuneata, Jatropha curcas, Lupinus angustifolius, Lupinus luteus, Lupinus mutabilis, Lespedeza sericea, Lespedeza striata, Lotus uliginosus, Lathyrus sativus, Lens culinaris, Lespedeza stipulacea, Linum usitatissimum, Lotus comiculatus, Lupinus albus, Lupinus angustifolius, Lupinus luteus, Medicago arborea, Medicago falcate, Medicago hispida, Medicago officinalis, Medicago. sativa Alfalfa, Medicago tribuloides, Macadamia integrifolia, Medicago arabica, Melilotus albus, Millettia pinnata, Mucuna pruriens, Olea europaea, Onobrychis viciifolia, Ornithopus sativus, Phaseolus aureus, Prunus cerasifera, Prunus cerasus, Phaseolus coccineus, Prunus domestica, Phaseolus lunatus, Prunus. maheleb, Phaseolus mungo, Prunus. persica, Prunus. pseudocerasus, Phaseolus vulgaris, Papaver somniferum, Phaseolus acutifolius, Phoenix dactylifera, Pistacia vera, Pisum sativum, Prunus amygdalus, Prunus armeniaca, Pueraria thunbergiana, Ribes nigrum, Ribes rubrum, Ribes grossularia, Ricinus communis, Sesamum indicum, Solanum tuberosum, Trifolium augustifolium, Trifolium diffusum, Trifolium hybridum, Trifolium incarnatum, Trifolium ingrescens, Trifolium pratense, Trifolium repens, Trifolium resupinatum, Trifolium subterraneum, Theobroma cacao, Trifolium alexandrinum, Trigonella foenumgraecum, Vemicia fordii, Vicia angustifolia, Vicia atropurpurea, Vicia calcarata, Vicia dasycarpa, Vicia ervilia, Vaccinium oxycoccos, Vicia pannonica, Vigna sesquipedalis, Vigna sinensis, Vicia villosa, Vicia faba, Vicia sative and Vigna angularis.

Plants and Plant Parts

In a further aspect the invention provides a plant that has increased root biomass as a result of having increased expression a PEAPOD protein, or fragment thereof.

In one embodiment expression of the PEAPOD protein, or fragment thereof, is increased as a consequence of the plant, or its ancestor plant or plant cell, having been transformed with a polynucleotide encoding the PEAPOD protein, or fragment thereof.

In a further embodiment plant is transgenic for a polynucleotide expressing the PEAPOD protein, or fragment thereof.

In a further embodiment the polynucleotide or fragment thereof is operatively linked polynucleotide to a tissue-preferred promoter.

In a further embodiment the promoter is a root-preferred promoter.

In a further embodiment the promoter is a root-specific promoter.

In a further embodiment the PEAPOD protein is as herein defined.

In a further embodiment the polynucleotide, encoding the PEAPOD protein, is as herein defined.

In a further aspect the invention provides a cell, part, propagule or progeny of the plant that is transgenic for at least one of:

-   a) the polynucleotide, and -   b) the polynucleotide and operatively linked promoter.

DETAILED DESCRIPTION

In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

The term “comprising” as used in this specification means “consisting at least in part of”. When interpreting each statement in this specification that includes the term “comprising”, features other than that or those prefaced by the term may also be present. Related terms such as “comprise” and “comprises” are to be interpreted in the same manner.

Increased Root Biomass

A plant with “increased root biomass” produces more root biomass than does a control plant of the same type and age. Thus “increased” means increased relative to a control plant of the same type and age.

Preferably the plant with “increased root biomass” produces at least 10%, preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 100%, more preferably at least 150%, more preferably at least 200%, more preferably at least 300%, more preferably at least 400% more root biomass than does a control plant of the same type and age.

In one embodiment the plant with “increased root biomass” has at least one of: larger roots, more roots, or a more extensive root system, than does a control plant.

Root Biomass

The term root biomass refers to total mass of root tissue produced by the plant. This can be assessed by dry weight or wet weight.

Root

The term root as used herein means the same as standard usage of the term. The term root encompasses the primary root, secondary roots, adventitious roots, root branches and root hairs. Roots are generally below ground, but the term also encompasses aerial roots. In one embodiment the term root encompasses non-leaf, non-node bearing parts of the plant.

Increased Drought Tolerance

In one embodiment the plant with “increased root biomass” also has increased drought tolerance. Again “increased” means increased relative to a control plant of the same type and age.

The term “increased drought tolerance” is intended to describe a plant, or plants, which perform more favourably in any aspect of their growth and development under sub-optimal hydration conditions than do suitable control plants in the same conditions.

Control Plant

In one embodiment the control plant is a wild-type plant. In a further embodiment the control plant is a non-transformed plant. In a further embodiment the control plant is a plant that has not been transformed with a PEAPOD polynucleotide. In a further embodiment the control plant is a plant that has not been transformed with a construct. In a further embodiment the control plant is a plant that has been transformed with a control construct. In one embodiment the construct is an empty vector construct.

Ectopic Expression

The term “ectopic expression” is intended to be interpreted broadly. The term refers to expression of a polynucleotide or polypeptide in any one of:

-   -   a cell, organ, tissue or plant where it is not normally         expressed,     -   a cell, organ, tissue or plant at a time, or developmental         stage, when it is not normally expressed, and     -   a cell, organ, tissue or plant at a level higher than it is         normally expressed in that cell, organ, tissue or plant.         Tissue Preferred Promoters

In certain embodiments, the PEAPOD protein encoding polynucleotides are expressed under the control of tissue preferred promoters. The term “preferred” with respect to tissue preferred promoters means that the promoter primarily drives expression in that tissue. Thus, for example, a root-preferred promoter drives a higher level of expression of an operably linked polynucleotide in root tissue than it does in other tissues or organs or the plant.

Root Preferred Promoters

A root-preferred promoter drives a higher level of expression of an operably linked polynucleotide in root tissue than it does in other tissues or organs or the plant.

Root-preferred promoters may include non-photosynthetic tissue preferred promoters and light-repressed regulated promoters.

Non-photosynthetic Tissue Preferred Promoters

Non-photosynthetic tissue preferred promoters include those preferentially expressed in non-photosynthetic tissues/organs of the plant.

Non-photosynthetic tissue preferred promoters may also include light repressed promoters.

Light Repressed Promoters

An example of a light repressed promoter is found in U.S. Pat. No. 5,639,952 and in U.S. Pat. No. 5,656,496.

Root Specific Promoters

An example of a root specific promoter is found in U.S. Pat. No. 5,837,848; and US 2004/0067506 and US 2001/0047525.

The term “preferentially expressed” with respect to a promoter being preferentially expressed in a certain tissue, means that the promoter is expressed at a higher level in that tissue than in other tissues of the plant.

The term “tissue specific” with respect to a promoter, means that the promoter is expressed substantially only in that tissue, and not other tissues of the plant.

In one embodiment the root-preferred promoter is a root-specific promoter.

The term “gene” as used herein means an endogenous genomic sequence which includes a coding sequence which encodes a polypeptide or protein. The coding sequence may be interrupted by one or more introns. A gene typically also includes a promoter sequence, 5′ untranslated sequence, 3′ untranslated sequence, and a terminator sequence. Genomic sequences that regulate expression of the protein may also be considered part of the gene.

Polynucleotides and Fragments

The term “polynucleotide(s),” as used herein, means a single or double-stranded deoxyribonucleotide or ribonucleotide polymer of any length but preferably at least 15 nucleotides, and include as non-limiting examples, coding and non-coding sequences of a gene, sense and antisense sequences complements, exons, introns, genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polypeptides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers and fragments.

A “fragment” of a polynucleotide refers to a contiguous subsequence of larger a polynucleotide sequence. Preferably the fragment is at least 15 nucleotides preferably at least 16 nucleotides, more preferably at least 17 nucleotides, more preferably at least 18 nucleotides, more preferably at least 19 nucleotides, more preferably at least 20 nucleotides, more preferably at least 21 nucleotides, more preferably at least 22 nucleotides, more preferably at least 23 nucleotides, more preferably at least 24 nucleotides, more preferably at least 25 nucleotides, more preferably at least 26 nucleotides, more preferably at least 27 nucleotides, more preferably at least 28 nucleotides, more preferably at least 29 nucleotides, more preferably at least 30 nucleotides, more preferably at least 31 nucleotides, more preferably at least 32 nucleotides, more preferably at least 33 nucleotides, more preferably at least 34 nucleotides, more preferably at least 35 nucleotides, more preferably at least 36 nucleotides, more preferably at least 37 nucleotides, more preferably at least 38 nucleotides, more preferably at least 39 nucleotides, more preferably at least 40 nucleotides, more preferably at least 41 nucleotides, more preferably at least 42 nucleotides, more preferably at least 43 nucleotides, more preferably at least 44 nucleotides, more preferably at least 45 nucleotides, more preferably at least 46 nucleotides, more preferably at least 47 nucleotides, more preferably at least 48 nucleotides, more preferably at least 49 nucleotides, more preferably at least 50 nucleotides, more preferably at least 51 nucleotides, more preferably at least 52 nucleotides, more preferably at least 53 nucleotides, more preferably at least 54 nucleotides, more preferably at least 55 nucleotides, more preferably at least 56 nucleotides, more preferably at least 57 nucleotides, more preferably at least 58 nucleotides, more preferably at least 59 nucleotides, more preferably at least 60 nucleotides, more preferably at least 61 nucleotides, more preferably at least 62 nucleotides, more preferably at least 63 nucleotides, more preferably at least 64 nucleotides, more preferably at least 65 nucleotides, more preferably at least 66 nucleotides, more preferably at least 67 nucleotides, more preferably at least 68 nucleotides, more preferably at least 69 nucleotides, more preferably at least 70 nucleotides, more preferably at least 71 nucleotides, more preferably at least 72 nucleotides, more preferably at least 73 nucleotides, more preferably at least 74 nucleotides, more preferably at least 75 nucleotides, more preferably at least 76 nucleotides, more preferably at least 77 nucleotides, more preferably at least 78 nucleotides, more preferably at least 79 nucleotides, more preferably at least 80 nucleotides, more preferably at least 81 nucleotides, more preferably at least 82 nucleotides, more preferably at least 83 nucleotides, more preferably at least 84 nucleotides, more preferably at least 85 nucleotides, more preferably at least 86 nucleotides, more preferably at least 87 nucleotides, more preferably at least 88 nucleotides, more preferably at least 89 nucleotides, more preferably at least 90 nucleotides, more preferably at least 91 nucleotides, more preferably at least 92 nucleotides, more preferably at least 93 nucleotides, more preferably at least 94 nucleotides, more preferably at least 95 nucleotides, more preferably at least 96 nucleotides, more preferably at least 97 nucleotides, more preferably at least 98 nucleotides, more preferably at least 99 nucleotides, more preferably at least 100 nucleotides, more preferably at least 150 nucleotides, more preferably at least 200 nucleotides, more preferably at least 250 nucleotides, more preferably at least 300 nucleotides, more preferably at least 350 nucleotides, more preferably at least 400 nucleotides, more preferably at least 450 nucleotides and most preferably at least 500 nucleotides of contiguous nucleotides of a polynucleotide disclosed. A fragment of a polynucleotide sequence can be used in antisense, RNA interference (RNAi), gene silencing, triple helix or ribozyme technology, or as a primer, a probe, included in a microarray, or used in polynucleotide-based selection methods of the invention.

In one embodiment the fragment encodes a polypeptide that performs, or is capable of performing, the same function as the polypeptide encoded by the larger polynucleotide that the fragment is part of.

The term “primer” refers to a short polynucleotide, usually having a free 3′OH group that is, or can be, hybridized to a template and used for priming polymerization of a polynucleotide complementary to the target.

The term “probe” refers to a short polynucleotide that is, or can be, used to detect a polynucleotide sequence that is complementary to the probe, in a hybridization-based assay. The probe may consist of a “fragment” of a polynucleotide as defined herein.

Polypeptides and Fragments

The term “polypeptide”, as used herein, encompasses amino acid chains of any length but preferably at least 5 amino acids, including full-length proteins, in which amino acid residues are linked by covalent peptide bonds. Polypeptides of the present invention, or used in the methods of the invention, may be purified natural products, or may be produced partially or wholly using recombinant or synthetic techniques. The term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or derivative thereof.

A “fragment” of a polynucleotide refers to a contiguous subsequence of a larger polynucleotide sequence. Preferably the fragment

A “fragment” of a polypeptide refers to a contiguous subsequence of larger a polypeptide. Preferably the fragment is at least 5, more preferably at least 10, more preferably at least 20, more preferably at least 30, more preferably at least 40, more preferably at least 50, more preferably at least 100, more preferably at least 120, more preferably at least 150, more preferably at least 200, more preferably at least 250, more preferably at least 300, more preferably at least 350, more preferably at least 400.

In one embodiment the fragment performs, or is capable of performing, the same function as the polypeptide that the fragment is part of.

Preferably the fragment performs a function that is required for the biological activity and/or provides three dimensional structure of the polypeptide.

The term “isolated” as applied to the polynucleotide or polypeptide sequences disclosed herein is used to refer to sequences that are removed from their natural cellular environment. In one embodiment the sequence is separated from its flanking sequences as found in nature. An isolated molecule may be obtained by any method or combination of methods including biochemical, recombinant, and synthetic techniques.

The term “recombinant” refers to a polynucleotide sequence that is synthetically produced or is removed from sequences that surround it in its natural context. The recombinant sequence may be recombined with sequences that are not present in its natural context.

A “recombinant” polypeptide sequence is produced by translation from a “recombinant” polynucleotide sequence.

The term “derived from” with respect to polynucleotides or polypeptides of the invention being derived from a particular genera or species, means that the polynucleotide or polypeptide has the same sequence as a polynucleotide or polypeptide found naturally in that genera or species. The polynucleotide or polypeptide, derived from a particular genera or species, may therefore be produced synthetically or recombinantly.

Variants

As used herein, the term “variant” refers to polynucleotide or polypeptide sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variants may be from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, variants of the polypeptides and polynucleotides disclosed herein possess biological activities that are the same or similar to those of the disclosed polypeptides or polypeptides. The term “variant” with reference to polypeptides and polynucleotides encompasses all forms of polypeptides and polynucleotides as defined herein.

Polynucleotide Variants

Variant polynucleotide sequences preferably exhibit at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, more preferably at least 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% identity to a sequence of the present invention. Identity is found over a comparison window of at least 20 nucleotide positions, preferably at least 50 nucleotide positions, more preferably at least 100 nucleotide positions, and most preferably over the entire length of a polynucleotide of the invention.

Polynucleotide sequence identity can be determined in the following manner. The subject polynucleotide sequence is compared to a candidate polynucleotide sequence using BLASTN (from the BLAST suite of programs, version 2.2.5[November 2002]) in bl2seq (Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences—a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250), which is publicly available from NCBI (ftp<dot>ncbi<dot>nih<dot>gov/blast/). In one embodiment the default parameters of bl2seq are utilized. In a further except the default parameters of bl2seq are utilized, except that filtering of low complexity parts should be turned off.

Polynucleotide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs (e.g. Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). A full implementation of the Needleman-Wunsch global alignment algorithm is found in the needle program in the EMBOSS package (Rice, P. Longden, I. and Bleasby, A. EMBOSS: The European Molecular Biology Open Software Suite, Trends in Genetics June 2000, vol 16, No 6. pp.276-277) which can be obtained from www<dot>hgmp<dot>mrc<dot>ac<dot>uk/Software/EMBOSS/. The European Bioinformatics Institute server also provides the facility to perform EMBOSS-needle global alignments between two sequences on line at www<dot>ebi<dot>ac<dot>uk/emboss/align/.

Alternatively the GAP program may be used which computes an optimal global alignment of two sequences without penalizing terminal gaps. GAP is described in the following paper: Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.

A preferred method for calculating polynucleotide % sequence identity is based on aligning sequences to be compared using Clustal X (Jeanmougin et al., 1998, Trends Biochem. Sci. 23, 403-5.)

Polynucleotide variants of the present invention also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance. Such sequence similarity with respect to polypeptides may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [November 2002]) from NCBI (ftp<dot>ncbi<dot>nih<dot>gov/blast/).

Alternatively, variant polynucleotides of the present invention hybridize to the specified polynucleotide sequences, or complements thereof under stringent conditions.

The term “hybridize under stringent conditions”, and grammatical equivalents thereof, refers to the ability of a polynucleotide molecule to hybridize to a target polynucleotide molecule (such as a target polynucleotide molecule immobilized on a DNA or RNA blot, such as a Southern blot or Northern blot) under defined conditions of temperature and salt concentration. The ability to hybridize under stringent hybridization conditions can be determined by initially hybridizing under less stringent conditions then increasing the stringency to the desired stringency.

With respect to polynucleotide molecules greater than about 100 bases in length, typical stringent hybridization conditions are no more than 25 to 30° C. (for example, 10° C.) below the melting temperature (Tm) of the native duplex (see generally, Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Ausubel et al., 1987, Current Protocols in Molecular Biology, Greene Publishing). Tm for polynucleotide molecules greater than about 100 bases can be calculated by the formula Tm=81. 5+0. 41% (G+C-log (Na+). (Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Bolton and McCarthy, 1962, PNAS 84:1390). Typical stringent conditions for polynucleotide of greater than 100 bases in length would be hybridization conditions such as prewashing in a solution of 6×SSC, 0.2% SDS; hybridizing at 65° C., 6×SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in 1×SSC, 0.1% SDS at 65° C. and two washes of 30 minutes each in 0.2×SSC, 0.1% SDS at 65° C.

With respect to polynucleotide molecules having a length less than 100 bases, exemplary stringent hybridization conditions are 5 to 10° C. below Tm. On average, the Tm of a polynucleotide molecule of length less than 100 bp is reduced by approximately (500/oligonucleotide length)° C.

With respect to the DNA mimics known as peptide nucleic acids (PNAs) (Nielsen et al., Science. 1991 Dec. 6; 254(5037):1497-500) Tm values are higher than those for DNA-DNA or DNA-RNA hybrids, and can be calculated using the formula described in Giesen et al., Nucleic Acids Res. 1998 Nov. 1; 26(21):5004-6. Exemplary stringent hybridization conditions for a DNA-PNA hybrid having a length less than 100 bases are 5 to 10° C. below the Tm.

Variant polynucleotides of the present invention also encompasses polynucleotides that differ from the sequences of the invention but that, as a consequence of the degeneracy of the genetic code, encode a polypeptide having similar activity to a polypeptide encoded by a polynucleotide of the present invention. A sequence alteration that does not change the amino acid sequence of the polypeptide is a “silent variation”. Except for ATG (methionine) and TGG (tryptophan), other codons for the same amino acid may be changed by art recognized techniques, e.g., to optimize codon expression in a particular host organism.

Polynucleotide sequence alterations resulting in conservative substitutions of one or several amino acids in the encoded polypeptide sequence without significantly altering its biological activity are also included in the invention. A skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie et al., 1990, Science 247, 1306).

Variant polynucleotides due to silent variations and conservative substitutions in the encoded polypeptide sequence may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [November 2002]) from NCBI (ftp<dot>ncbi<dot>nih<dot>gov/blast/) via the tblastx algorithm as previously described.

Polypeptide Variants

The term “variant” with reference to polypeptides encompasses naturally occurring, recombinantly and synthetically produced polypeptides. Variant polypeptide sequences preferably exhibit at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, more preferably at least 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% identity to a sequences of the present invention. Identity is found over a comparison window of at least 20 amino acid positions, preferably at least 50 amino acid positions, more preferably at least 100 amino acid positions, and most preferably over the entire length of a polypeptide of the invention.

Polypeptide sequence identity can be determined in the following manner. The subject polypeptide sequence is compared to a candidate polypeptide sequence using BLASTP (from the BLAST suite of programs, version 2.2.5 [November 2002]) in bl2seq, which is publicly available from NCBI (ftp<dot>ncbi<dot>nih<dot>gov/blast/). In one embodiment the default parameters of bl2seq are utilized. In a further except the default parameters of bl2seq are utilized, except that filtering of low complexity parts should be turned off.

Polypeptide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs. EMBOSS-needle (available www<dot>ebi<dot>ac<dot>uk/emboss/align/) and GAP (Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.) as discussed above are also suitable global sequence alignment programs for calculating polypeptide sequence identity.

A preferred method for calculating polypeptide % sequence identity is based on aligning sequences to be compared using Clustal X (Jeanmougin et al., 1998, Trends Biochem. Sci. 23, 403-5.)

A variant polypeptide includes a polypeptide wherein the amino acid sequence differs from a polypeptide herein by one or more conservative amino acid substitutions, deletions, additions or insertions which do not affect the biological activity of the peptide. Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g., substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagines, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

Non-conservative substitutions will entail exchanging a member of one of these classes for a member of another class.

Analysis of evolved biological sequences has shown that not all sequence changes are equally likely, reflecting at least in part the differences in conservative versus non-conservative substitutions at a biological level. For example, certain amino acid substitutions may occur frequently, whereas others are very rare. Evolutionary changes or substitutions in amino acid residues can be modelled by a scoring matrix also referred to as a substitution matrix. Such matrices are used in bioinformatics analysis to identify relationships between sequences, one example being the BLOSUM62 matrix shown below (Table 1).

TABLE 1 The BLOSUM62 matrix containing all possible substitution scores [Henikoff and Henikoff, 1992]. A R N D C Q E G H I L K M F P S T W T V A 4 −1 −2 −2 0 −1 −1 0 −2 −1 −1 −1 −1 −2 −1 1 0 −3 −2 0 R −1 5 0 −2 −3 1 0 −2 0 −3 −2 2 −1 −3 −2 −1 −1 −3 −2 −3 N −2 0 6 1 −3 0 0 0 1 −3 −3 0 −2 −3 −2 1 0 −4 −2 −3 D −2 −2 1 6 −3 0 2 −1 −1 −3 −4 −1 −3 −3 −1 0 −1 −4 −3 −3 C 0 −3 −3 −3 9 −3 −4 −3 −3 −1 −1 −3 −1 −2 −3 −1 −1 −2 −2 −1 Q −1 1 0 0 −3 5 2 −2 0 −3 −2 1 0 −3 −1 0 −1 −2 −1 −3 E −1 0 0 2 −4 3 5 −2 0 −3 −3 1 −2 −3 −1 0 −1 −3 −2 −2 G 0 −2 0 −1 −3 −2 −2 6 −2 −4 −4 −2 −3 −3 −2 0 −2 −2 −3 −3 H −2 0 1 −1 −3 0 0 −2 −3 −3 −3 −1 −2 −1 −2 −1 −2 −2 2 −3 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 2 −3 1 0 −3 −2 −1 −3 −1 3 L −1 −2 −3 −4 −1 −2 −3 −4 −3 2 −4 −2 2 0 −3 −2 −1 −2 −1 1 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 −1 −3 −1 0 −1 −3 −2 −2 M −1 −1 −2 −3 −1 0 −2 −3 −2 1 2 −1 5 0 −2 −1 −1 −1 −1 1 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 −4 −2 −2 1 3 −1 P −1 −2 −2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 −1 −1 −4 −3 −2 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1 −2 −1 4 1 −3 −2 −2 T 0 −1 0 −1 −1 −1 −1 −3 −2 −1 −1 −1 −1 −2 −1 1 5 −2 −2 0 W −3 −3 −4 −4 −2 −2 −3 −2 −2 −2 −2 −3 −1 1 −4 −3 −2 11 2 −3 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1 −1 −2 −1 3 −3 −2 −2 2 7 −1 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0 −3 −1 4

The BLOSUM62 matrix shown is used to generate a score for each aligned amino acid pair found at the intersection of the corresponding column and row. For example, the substitution score from a glutamic acid residue (E) to an aspartic acid residue (D) is 2. The diagonal show scores for amino acids which have not changed. Most substitutions changes have a negative score. The matrix contains only whole numbers.

Determination of an appropriate scoring matrix to produce the best alignment for a given set of sequences is believed to be within the skill of in the art. The BLOSUM62 matrix in table 1 is also used as the default matrix in BLAST searches, although not limited thereto.

Other variants include peptides with modifications which influence peptide stability. Such analogs may contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the peptide sequence. Also included are analogs that include residues other than naturally occurring L-amino acids, e.g. D-amino acids or non-naturally occurring synthetic amino acids, e.g. beta or gamma amino acids and cyclic analogs

Constructs, Vectors and Components thereof

The term “genetic construct” refers to a polynucleotide molecule, usually double-stranded DNA, which may have inserted into it another polynucleotide molecule (the insert polynucleotide molecule) such as, but not limited to, a cDNA molecule. A genetic construct may contain the necessary elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating the transcript into a polypeptide. The insert polynucleotide molecule may be derived from the host cell, or may be derived from a different cell or organism and/or may be a recombinant polynucleotide. Once inside the host cell the genetic construct may become integrated in the host chromosomal DNA. The genetic construct may be linked to a vector.

The term “vector” refers to a polynucleotide molecule, usually double stranded DNA, which is used to transport the genetic construct into a host cell. The vector may be capable of replication in at least one additional host system, such as E. coli.

The term “expression construct” refers to a genetic construct that includes the necessary elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating the transcript into a polypeptide. An expression construct typically comprises in a 5′ to 3′ direction:

-   -   a) a promoter functional in the host cell into which the         construct will be transformed,     -   b) the polynucleotide to be expressed, and     -   c) a terminator functional in the host cell into which the         construct will be transformed.

The term “coding region” or “open reading frame” (ORF) refers to the sense strand of a genomic DNA sequence or a cDNA sequence that is capable of producing a transcription product and/or a polypeptide under the control of appropriate regulatory sequences. The coding sequence is identified by the presence of a 5′ translation start codon and a 3′ translation stop codon. When inserted into a genetic construct, a “coding sequence” is capable of being expressed when it is operably linked to promoter and terminator sequences.

“Operably-linked” means that the sequenced to be expressed is placed under the control of regulatory elements that include promoters, tissue-specific regulatory elements, temporal regulatory elements, enhancers, repressors and terminators.

The term “noncoding region” refers to untranslated sequences that are upstream of the translational start site and downstream of the translational stop site. These sequences are also referred to respectively as the 5′ UTR and the 3′ UTR. These regions include elements required for transcription initiation and termination and for regulation of translation efficiency.

Terminators are sequences, which terminate transcription, and are found in the 3′ untranslated ends of genes downstream of the translated sequence. Terminators are important determinants of mRNA stability and in some cases have been found to have spatial regulatory functions.

The term “promoter” refers to non-transcribed cis-regulatory elements upstream of the coding region that regulate gene transcription. Promoters comprise cis-initiator elements which specify the transcription initiation site and conserved boxes such as the TATA box, and motifs that are bound by transcription factors.

A promoter may be homologous with respect to the polynucleotide to be expressed. This means that the promoter and polynucleotide are found operably linked in nature.

Alternatively the promoter may be heterologous with respect to the polynucleotide to be expressed. This means that the promoter and the polynucleotide are not found operably linked in nature.

A “transgene” is a polynucleotide that is introduced into an organism by transformation. The transgene may be derived from the same species or from a different species as the species of the organism into which the transgene is introduced. The transgene may also be synthetic and not found in nature in any species.

A “transgenic plant” refers to a plant which contains new genetic material as a result of genetic manipulation or transformation. The new genetic material may be derived from a plant of the same species as the resulting transgenic plant or from a different species, or may be synthetic.

Preferably the “transgenic plant” is different from any plant found in nature due the presence of the transgene.

An “inverted repeat” is a sequence that is repeated, where the second half of the repeat is in the complementary strand, e.g.,

(SEQ ID NO: 142) (5′)GATCTA TAGATC(3′) (SEQ ID NO: 143) (3′)CTAGAT ATCTAG(5′).

Read-through transcription will produce a transcript that undergoes complementary base-pairing to form a hairpin structure provided that there is a 3-5 bp spacer between the repeated regions. The spacer can be any polynucleotide sequence but is typically at least 3 base pairs in length.

Host Cells

Host cells may be derived from, for example, bacterial, fungal, insect, mammalian or plant organisms.

Methods for Isolating or Producing Polynucleotides

The polynucleotide molecules of the invention can be isolated by using a variety of techniques known to those of ordinary skill in the art. By way of example, such polypeptides can be isolated through use of the polymerase chain reaction (PCR) described in Mullis et al., Eds. 1994 The Polymerase Chain Reaction, Birkhauser, incorporated herein by reference. The polypeptides of the invention can be amplified using primers, as defined herein, derived from the polynucleotide sequences of the invention.

Further methods for isolating polynucleotides of the invention include use of all, or portions of, the polypeptides having the sequence set forth herein as hybridization probes. The technique of hybridizing labelled polynucleotide probes to polynucleotides immobilized on solid supports such as nitrocellulose filters or nylon membranes, can be used to screen the genomic or cDNA libraries. Exemplary hybridization and wash conditions are: hybridization for 20 hours at 65° C. in 5. 0×SSC, 0. 5% sodium dodecyl sulfate, 1×Denhardt's solution; washing (three washes of twenty minutes each at 55° C.) in 1. 0×SSC, 1% (w/v) sodium dodecyl sulfate, and optionally one wash (for twenty minutes) in 0. 5×SSC, 1% (w/v) sodium dodecyl sulfate, at 60° C. An optional further wash (for twenty minutes) can be conducted under conditions of 0. 1×SSC, 1% (w/v) sodium dodecyl sulfate, at 60° C.

The polynucleotide fragments of the invention may be produced by techniques well-known in the art such as restriction endonuclease digestion, oligonucleotide synthesis and PCR amplification.

A partial polynucleotide sequence may be used, in methods well-known in the art to identify the corresponding full length polynucleotide sequence. Such methods include PCR-based methods, 5′RACE (Frohman M A, 1993, Methods Enzymol. 218: 340-56) and hybridization-based method, computer/database-based methods. Further, by way of example, inverse PCR permits acquisition of unknown sequences, flanking the polynucleotide sequences disclosed herein, starting with primers based on a known region (Triglia et al., 1998, Nucleic Acids Res 16, 8186, incorporated herein by reference). The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template. Divergent primers are designed from the known region. In order to physically assemble full-length clones, standard molecular biology approaches can be utilized (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987).

It may be beneficial, when producing a transgenic plant from a particular species, to transform such a plant with a sequence or sequences derived from that species. The benefit may be to alleviate public concerns regarding cross-species transformation in generating transgenic organisms. Additionally when down-regulation of a gene is the desired result, it may be necessary to utilise a sequence identical (or at least highly similar) to that in the plant, for which reduced expression is desired. For these reasons among others, it is desirable to be able to identify and isolate orthologues of a particular gene in several different plant species.

Variants (including orthologues) may be identified by the methods described.

Methods for Identifying Variants

Physical Methods

Variant polypeptides may be identified using PCR-based methods (Mullis et al., Eds. 1994 The Polymerase Chain Reaction, Birkhauser). Typically, the polynucleotide sequence of a primer, useful to amplify variants of polynucleotide molecules of the invention by PCR, may be based on a sequence encoding a conserved region of the corresponding amino acid sequence.

Alternatively library screening methods, well known to those skilled in the art, may be employed (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987). When identifying variants of the probe sequence, hybridization and/or wash stringency will typically be reduced relatively to when exact sequence matches are sought.

Polypeptide variants may also be identified by physical methods, for example by screening expression libraries using antibodies raised against polypeptides of the invention (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987) or by identifying polypeptides from natural sources with the aid of such antibodies.

Computer Based Methods

The variant sequences of the invention, including both polynucleotide and polypeptide variants, may also be identified by computer-based methods well-known to those skilled in the art, using public domain sequence alignment algorithms and sequence similarity search tools to search sequence databases (public domain databases include Genbank, EMBL, Swiss-Prot, PIR and others). See, e.g., Nucleic Acids Res. 29: 1-10 and 11-16, 2001 for examples of online resources. Similarity searches retrieve and align target sequences for comparison with a sequence to be analyzed (i.e., a query sequence). Sequence comparison algorithms use scoring matrices to assign an overall score to each of the alignments.

An exemplary family of programs useful for identifying variants in sequence databases is the BLAST suite of programs (version 2.2.5 [November 2002]) including BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX, which are publicly available from (ftp<dot>ncbi<dot>nih<dot>gov/blast/) or from the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894 USA. The NCBI server also provides the facility to use the programs to screen a number of publicly available sequence databases. BLASTN compares a nucleotide query sequence against a nucleotide sequence database. BLASTP compares an amino acid query sequence against a protein sequence database. BLASTX compares a nucleotide query sequence translated in all reading frames against a protein sequence database. tBLASTN compares a protein query sequence against a nucleotide sequence database dynamically translated in all reading frames. tBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database. The BLAST programs may be used with default parameters or the parameters may be altered as required to refine the screen.

The use of the BLAST family of algorithms, including BLASTN, BLASTP, and BLASTX, is described in the publication of Altschul et al., Nucleic Acids Res. 25: 3389-3402, 1997.

The “hits” to one or more database sequences by a queried sequence produced by BLASTN, BLASTP, BLASTX, tBLASTN, tBLASTX, or a similar algorithm, align and identify similar portions of sequences. The hits are arranged in order of the degree of similarity and the length of sequence overlap. Hits to a database sequence generally represent an overlap over only a fraction of the sequence length of the queried sequence.

The BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX algorithms also produce “Expect” values for alignments. The Expect value (E) indicates the number of hits one can “expect” to see by chance when searching a database of the same size containing random contiguous sequences. The Expect value is used as a significance threshold for determining whether the hit to a database indicates true similarity. For example, an E value of 0.1 assigned to a polynucleotide hit is interpreted as meaning that in a database of the size of the database screened, one might expect to see 0.1 matches over the aligned portion of the sequence with a similar score simply by chance. For sequences having an E value of 0.01 or less over aligned and matched portions, the probability of finding a match by chance in that database is 1% or less using the BLASTN, BLASTP, BLASTX, tBLASTN or tBLASTX algorithm.

Multiple sequence alignments of a group of related sequences can be carried out with CLUSTALW (Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22:4673-4680, www-igbmc<dot>u-strasbq<dot>fr/Biolnfo/ClustalW/Top<dot>html) or T-COFFEE (Cedric Notredame, Desmond G. Higgins, Jaap Heringa, T-Coffee: A novel method for fast and accurate multiple sequence alignment, J. Mol. Biol. (2000) 302: 205-217)) or PILEUP, which uses progressive, pairwise alignments. (Feng and Doolittle, 1987, J. Mol. Evol. 25, 351).

Pattern recognition software applications are available for finding motifs or signature sequences. For example, MEME (Multiple Em for Motif Elicitation) finds motifs and signature sequences in a set of sequences, and MAST (Motif Alignment and Search Tool) uses these motifs to identify similar or the same motifs in query sequences. The MAST results are provided as a series of alignments with appropriate statistical data and a visual overview of the motifs found. MEME and MAST were developed at the University of California, San Diego.

PROSITE (Bairoch and Bucher, 1994, Nucleic Acids Res. 22, 3583; Hofmann et al., 1999, Nucleic Acids Res. 27, 215) is a method of identifying the functions of uncharacterized proteins translated from genomic or cDNA sequences. The PROSITE database (www<dot>expasy<dot>org/prosite) contains biologically significant patterns and profiles and is designed so that it can be used with appropriate computational tools to assign a new sequence to a known family of proteins or to determine which known domain(s) are present in the sequence (Falquet et al., 2002, Nucleic Acids Res. 30, 235). Prosearch is a tool that can search SWISS-PROT and EMBL databases with a given sequence pattern or signature.

Methods for Isolating Polypeptides

The polypeptides of the invention, or used in the methods of the invention, including variant polypeptides, may be prepared using peptide synthesis methods well known in the art such as direct peptide synthesis using solid phase techniques (e.g. Stewart et al., 1969, in Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco Calif., or automated synthesis, for example using an Applied Biosystems 431A Peptide Synthesizer (Foster City, Calif.). Mutated forms of the polypeptides may also be produced during such syntheses.

The polypeptides and variant polypeptides of the invention, or used in the methods of the invention, may also be purified from natural sources using a variety of techniques that are well known in the art (e.g. Deutscher, 1990, Ed, Methods in Enzymology, Vol. 182, Guide to Protein Purification).

Alternatively the polypeptides and variant polypeptides of the invention, or used in the methods of the invention, may be expressed recombinantly in suitable host cells and separated from the cells as discussed below.

Methods for Producing Constructs and Vectors

The genetic constructs of the present invention comprise one or more polynucleotide sequences of the invention and/or polynucleotides encoding polypeptides of the invention, and may be useful for transforming, for example, bacterial, fungal, insect, mammalian or plant organisms. The genetic constructs of the invention are intended to include expression constructs as herein defined.

Methods for producing and using genetic constructs and vectors are well known in the art and are described generally in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987).

Methods for Producing Host Cells Comprising Polynucleotides, Constructs or Vectors

The invention provides a host cell which comprises a genetic construct or vector of the invention.

Host cells comprising genetic constructs, such as expression constructs, of the invention are useful in methods well known in the art (e.g. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987) for recombinant production of polypeptides of the invention. Such methods may involve the culture of host cells in an appropriate medium in conditions suitable for or conducive to expression of a polypeptide of the invention. The expressed recombinant polypeptide, which may optionally be secreted into the culture, may then be separated from the medium, host cells or culture medium by methods well known in the art (e.g. Deutscher, Ed, 1990, Methods in Enzymology, Vol 182, Guide to Protein Purification).

Methods for Producing Plant Cells and Plants Comprising Constructs and Vectors

The invention further provides plant cells which comprise a genetic construct of the invention, and plant cells modified to alter expression of a polynucleotide or polypeptide of the invention, or used in the methods of the invention. Plants comprising such cells also form an aspect of the invention.

Methods for transforming plant cells, plants and portions thereof with polypeptides are described in Draper et al., 1988, Plant Genetic Transformation and Gene Expression. A Laboratory Manual. Blackwell Sci. Pub. Oxford, p. 365; Potrykus and Spangenburg, 1995, Gene Transfer to Plants. Springer-Verlag, Berlin.; and Gelvin et al., 1993, Plant Molecular Biol. Manual. Kluwer Acad. Pub. Dordrecht. A review of transgenic plants, including transformation techniques, is provided in Galun and Breiman, 1997, Transgenic Plants. Imperial College Press, London.

Methods for Genetic Manipulation of Plants

A number of plant transformation strategies are available (e.g. Birch, 1997, Ann Rev Plant Phys Plant Mol Biol, 48, 297, Hellens R P, et al (2000) Plant Mol Biol 42: 819-32, Hellens R et al Plant Meth 1: 13). For example, strategies may be designed to increase expression of a polynucleotide/polypeptide in a plant cell, organ and/or at a particular developmental stage where/when it is normally expressed or to ectopically express a polynucleotide/polypeptide in a cell, tissue, organ and/or at a particular developmental stage which/when it is not normally expressed. The expressed polynucleotide/polypeptide may be derived from the plant species to be transformed or may be derived from a different plant species.

Genetic constructs for expression of genes in transgenic plants typically include promoters for driving the expression of one or more cloned polynucleotide, terminators and selectable marker sequences to detect presence of the genetic construct in the transformed plant.

The promoters suitable for use in genetic constructs may be functional in a cell, tissue or organ of a monocot or dicot plant and include cell-, tissue- and organ-specific promoters, cell cycle specific promoters, temporal promoters, inducible promoters, constitutive promoters that are active in most plant tissues, and recombinant promoters. Choice of promoter will depend upon the temporal and spatial expression of the cloned polynucleotide, so desired. The promoters may be those normally associated with a transgene of interest, or promoters which are derived from genes of other plants, viruses, and plant pathogenic bacteria and fungi. Those skilled in the art will, without undue experimentation, be able to select promoters that are suitable for use in modifying and modulating plant traits using genetic constructs comprising the polynucleotide sequences of the invention. Examples of constitutive plant promoters include the CaMV 35S promoter, the nopaline synthase promoter and the octopine synthase promoter, and the Ubi 1 promoter from maize. Plant promoters which are active in specific tissues, respond to internal developmental signals or external abiotic or biotic stresses are described in the scientific literature. Exemplary promoters are described, e.g., in WO 02/00894, which is herein incorporated by reference.

Exemplary terminators that are commonly used in plant transformation genetic construct include, e.g., the cauliflower mosaic virus (CaMV) 35S terminator, the Agrobacterium tumefaciens nopaline synthase or octopine synthase terminators, the Zea mays zein gene terminator, the Oryza sativa ADP-glucose pyrophosphorylase terminator and the Solanum tuberosum PI-II terminator.

Selectable markers commonly used in plant transformation include the neomycin phophotransferase II gene (NPT II) which confers kanamycin resistance, the aadA gene, which confers spectinomycin and streptomycin resistance, the phosphinothricin acetyl transferase (bar gene) for Ignite (AgrEvo) and Basta (Hoechst) resistance, and the hygromycin phosphotransferase gene (hpt) for hygromycin resistance.

Use of genetic constructs comprising reporter genes (coding sequences which express an activity that is foreign to the host, usually an enzymatic activity and/or a visible signal (e.g., luciferase, GUS, GFP) which may be used for promoter expression analysis in plants and plant tissues are also contemplated. The reporter gene literature is reviewed in Herrera-Estrella et al., 1993, Nature 303, 209, and Schrott, 1995, In: Gene Transfer to Plants (Potrykus, T., Spangenberg. Eds) Springer Verlag. Berline, pp. 325-336.

Gene Silencing

Transformation strategies may be designed to reduce expression of a polynucleotide/polypeptide in a plant cell, tissue, organ or at a particular developmental stage which/when it is normally expressed. Such strategies are known as gene silencing strategies.

Gene silencing strategies may be focused on the gene itself or regulatory elements which effect expression of the encoded polypeptide. “Regulatory elements” is used here in the widest possible sense and includes other genes which interact with the gene of interest.

Genetic constructs designed to decrease or silence the expression of a polynucleotide/polypeptide of the invention may include an antisense copy of a polynucleotide of the invention. In such constructs the polynucleotide is placed in an antisense orientation with respect to the promoter and terminator.

An “antisense” polynucleotide is obtained by inverting a polynucleotide or a segment of the polynucleotide so that the transcript produced will be complementary to the mRNA transcript of the gene, e.g.,

5′GATCTA 3′ (coding 3′CTAGAT 5′ (antisense strand) strand) 3′CUAGAU 5′ mRNA 5′GAUCUCG 3′ antisense RNA

Genetic constructs designed for gene silencing may also include an inverted repeat. An ‘inverted repeat’ is a sequence that is repeated where the second half of the repeat is in the complementary strand, e.g.,

(SEQ ID NO: 144) 5′-GATCTA . . . TAGATC-3′ (SEQ ID NO: 145) 3′-CTAGAT . . . ATCTAG-5′.

The transcript formed may undergo complementary base pairing to form a hairpin structure. Usually a spacer of at least 3-5 bp between the repeated region is required to allow hairpin formation. Constructs including such invented repeat sequences may be used in RNA interference (RNAi) and therefore can be referred to as RNAi constructs.

Another silencing approach involves the use of a small antisense RNA targeted to the transcript equivalent to an miRNA (Llave et al., 2002, Science 297, 2053). Use of such small antisense RNA corresponding to polynucleotide of the invention is expressly contemplated.

The term genetic construct as used herein also includes small antisense RNAs and other such polypeptides effecting gene silencing.

Transformation with an expression construct, as herein defined, may also result in gene silencing through a process known as sense suppression (e.g. Napoli et al., 1990, Plant Cell 2, 279; de Carvalho Niebel et al., 1995, Plant Cell, 7, 347). In some cases sense suppression may involve over-expression of the whole or a partial coding sequence but may also involve expression of non-coding region of the gene, such as an intron or a 5′ or 3′ untranslated region (UTR). Chimeric partial sense constructs can be used to coordinately silence multiple genes (Abbott et al., 2002, Plant Physiol. 128(3): 844-53; Jones et al., 1998, Planta 204: 499-505). The use of such sense suppression strategies to silence the expression of a polynucleotide of the invention is also contemplated.

The polynucleotide inserts in genetic constructs designed for gene silencing may correspond to coding sequence and/or non-coding sequence, such as promoter and/or intron and/or 5′ or 3′ UTR sequence, of the corresponding gene.

Other gene silencing strategies include dominant negative approaches and the use of ribozyme constructs (McIntyre, 1996, Transgenic Res, 5, 257).

Pre-transcriptional silencing may be brought about through mutation of the gene itself or its regulatory elements. Such mutations may include point mutations, frameshifts, insertions, deletions and substitutions.

Transformation Protocols

The following are representative publications disclosing genetic transformation protocols that can be used to genetically transform the following plant species: Rice (Alam et al., 1999, Plant Cell Rep. 18, 572); apple (Yao et al., 1995, Plant Cell Reports 14, 407-412); maize (U.S. Pat. Nos. 5,177,010 and 5,981,840); wheat (Ortiz et al., 1996, Plant Cell Rep. 15, 1996, 877); tomato (U.S. Pat. No. 5,159,135); potato (Kumar et al., 1996 Plant J. 9, : 821); cassava (Li et al., 1996 Nat. Biotechnology 14, 736); lettuce (Michelmore et al., 1987, Plant Cell Rep. 6, 439); tobacco (Horsch et al., 1985, Science 227, 1229); cotton (U.S. Pat. Nos. 5,846,797 and 5,004,863); grasses (U.S. Pat. Nos. 5,187,073 and 6,020,539); peppermint (Niu et al., 1998, Plant Cell Rep. 17, 165); citrus plants (Pena et al., 1995, Plant Sci. 104, 183); caraway (Krens et al., 1997, Plant Cell Rep, 17, 39); banana (U.S. Pat. No. 5,792,935); soybean (U.S. Pat. Nos. 5,416,011; 5,569,834; 5,824,877; 5,563,04455 and 5,968,830); pineapple (U.S. Pat. No. 5,952,543); poplar (U.S. Pat. No. 4,795,855); monocots in general (U.S. Pat. Nos. 5,591,616 and 6,037,522); brassica (U.S. Pat. Nos. 5,188,958; 5,463,174 and 5,750,871); cereals (U.S. Pat. No. 6,074,877); pear (Matsuda et al., 2005, Plant Cell Rep. 24(1):45-51); Prunus (Ramesh et al., 2006 Plant Cell Rep. 25(8):821-8; Song and Sink 2005 Plant Cell Rep. 2006; 25(2):117-23; Gonzalez Padilla et al., 2003 Plant Cell Rep. 22(1):38-45); strawberry (Oosumi et al., 2006 Planta. 223(6):1219-30; Folta et al., 2006 Planta April 14; PMID: 16614818), rose (Li et al., 2003), Rubus (Graham et al., 1995 Methods Mol Biol. 1995; 44:129-33), tomato (Dan et al., 2006, Plant Cell Reports V25:432-441), apple (Yao et al., 1995, Plant Cell Rep. 14, 407-412) and Actinidia eriantha (Wang et al., 2006, Plant Cell Rep. 25, 5: 425-31), silver birch (Keinonen-Mettala et al., 1998, Plant Cell Rep. 17: 356-361.) and aspen (Nilsson O, et al., 1992, Transgenic Research. 1: 209-220). Transformation of other species is also contemplated by the invention. Suitable methods and protocols are available in the scientific literature.

Several further methods known in the art may be employed to alter expression of activity of a nucleotide and/or polypeptide of the invention. Such methods include but are not limited to Tilling (Till et al., 2003, Methods Mol Biol, 2%, 205), so called “Deletagene” technology (Li et al., 2001, Plant Journal 27(3), 235) and the use of artificial transcription factors such as synthetic zinc finger transcription factors. (e.g. Jouvenot et al., 2003, Gene Therapy 10, 513). Additionally antibodies or fragments thereof, targeted to a particular polypeptide may also be expressed in plants to modulate the activity of that polypeptide (Jobling et al., 2003, Nat. Biotechnol., 21(1), 35). Transposon tagging approaches may also be applied. Additionally peptides interacting with a polypeptide of the invention may be identified through technologies such as phase-display (Dyax Corporation). Such interacting peptides may be expressed in or applied to a plant to affect activity of a polypeptide of the invention. Use of each of the above approaches in alteration of expression of a nucleotide and/or polypeptide of the invention is specifically contemplated.

The terms “to alter expression of” and “altered expression” of a polynucleotide or polypeptide of the invention, or used in the methods of the invention, are intended to encompass the situation where genomic DNA corresponding to a polynucleotide of the invention is modified thus leading to altered expression of a polynucleotide or polypeptide of the invention. Modification of the genomic DNA may be through genetic transformation or other methods known in the art for inducing mutations. The “altered expression” can be related to an increase or decrease in the amount of messenger RNA and/or polypeptide produced and may also result in altered activity of a polypeptide due to alterations in the sequence of a polynucleotide and polypeptide produced.

Methods of Selecting Plants

Methods are also provided for selecting plants with increased root biomass. Such methods involve testing of plants for altered for the expression of at least one PEAPOD polynucleotide or polypeptide, including those as defined or disclosed herein. Such methods may be applied at a young age or early developmental stage when the increased root biomass characteristics may not necessarily be easily measurable.

The expression of a polynucleotide, such as a messenger RNA, is often used as an indicator of expression of a corresponding polypeptide. Exemplary methods for measuring the expression of a polynucleotide include but are not limited to Northern analysis, RT-PCR and dot-blot analysis (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987). Polynucleotides or portions of the polynucleotides of the invention are thus useful as probes or primers, as herein defined, in methods for the identification of plants with increased root biomass. The polynucleotides of the invention, or disclosed herein, may be used as probes in hybridization experiments, or as primers in PCR based experiments, designed to identify such plants.

Alternatively antibodies may be raised against PEAPOD polypeptides as described or disclosed herein. Methods for raising and using antibodies are standard in the art (see for example: Antibodies, A Laboratory Manual, Harlow A Lane, Eds, Cold Spring Harbour Laboratory, 1998). Such antibodies may be used in methods to detect altered expression of such polypeptides. Such methods may include ELISA (Kemeny, 1991, A Practical Guide to ELISA, NY Pergamon Press) and Western analysis (Towbin & Gordon, 1994, J Immunol Methods, 72, 313).

These approaches for analysis of polynucleotide or polypeptide expression and the selection of plants with increased root biomass are useful in conventional breeding programs designed to produce varieties with such altered characteristics.

Plants

The term “plant” is intended to include a whole plant, any part of a plant, propagules and progeny of a plant.

The term ‘propagule’ means any part of a plant that may be used in reproduction or propagation, either sexual or asexual, including seeds and cuttings.

The plants of the invention may be grown and either self-ed or crossed with a different plant strain and the resulting hybrids, with the desired phenotypic characteristics, may be identified. Two or more generations may be grown to ensure that the subject phenotypic characteristics are stably maintained and inherited. Plants resulting from such standard breeding approaches also form an aspect of the present invention.

Control of Plant Growth and Development by Gibberellins (GA), Brassinosteroids (BR) and other Plant Hormones

Gibberellins (GA) and Brassinosteroids (BR) are two classes of plant hormones; between them they are involved in many aspects of plant morphogenesis and growth; including: seed germination, cell elongation, vascular development, see size, leaf erectness, flowering, leaf and fruit senescence (Mathew et al 2009, NZJAR 52, 213-225; Hou et al 2010, Developmental Cell 19, 884-894; Jiang and Lin 2013, Plant Signaling and Behaviour 8:10, e25928).

Given their roles in plant development the ability to manipulate either the levels of GA and BR or their downstream targets is highly desirable in terms of improving both yield and quality in many plant species. Indeed there are some commercial examples where exogenous applications of either hormone are used to improve agronomic value.

GA can be applied to ryegrass pasture to stimulate out-of-season growth as well as promote flowering (Mathew et al 2009, NZJAR 52, 213-225), it can also be used to counteract the adverse effects of cooler temperatures on sugarcane (a tropical C4 grass). GAs are also used to enlarge fruit size of seedless grapes and cherries, to promote fruit set in apple and pear and to delay rind-aging in particular citrus crops (Sun 2011, Current Biology 21, R338-R345). Similarly, BR preparations are recommended for improving crop yield and quality of tomato, potato, cucumber, pepper and barley, rice, maize, wheat, cotton, and tobacco (Prusakova et al 1999, Agrarian Russia, 41-44; Khripach et al 2000, Annals of Botany 86, 441-447; Anjum et al 2011, J. Agronomy Crop Sci. 197, 177-185; Vardhini 2012, J. Phytology 4, 1-3). However, the low adoption of commercially applied brassinosteroids may reflect the cost and the fact that plants do not efficiently absorb steroids when they are applied exogenously. In addition, the need to strictly control timing and concentration of exogenous supplied GA and BR limits their applications.

For the most part the GA and BR biosynthesis and catabolic pathways in angiosperms have been characterized and include negative regulators and downstream transcription factor targets. Upon binding GA or BR to their respective receptor a complex signal pathway ensues and in both cases a central point of regulation involves the ubiquitin—proteasome pathway altering the level of the negative regulator DELLA (in the case of GA) and the transcriptional regulator BZR1 (in the case of BR).

The removal of DELLA proteins results in the removal of growth repression and promotion of GA-responsive growth and development. Conversely the detection of BR leads to the accumulation of unphosphorylated BZR1 protein in the nucleus. Dephosphorylation of BZR1 prevents its degradation by the proteasome and instead allows the binding of BZR1 with other DNA binding transcription factors and interacts with transcriptional cofactors. This leads to the regulation of thousands of genes involved in growth and other cellular processes, including the inhibition of expression of BR biosynthetic genes (He et al 2005, Science 307, 1634-1638; Guo et al 2013, Current Opinion Plant Biol. 16, 545-553).

There are a number of endogenous signals and environmental cues that influence the GA-GID1-DELLA regulatory module in which DELLA integrates different signalling activities by direct protein-protein interaction with multiple key regulatory proteins from other pathways. As such DELLA proteins are master growth repressors that control plant growth and development by integrating internal signals from other hormone pathways (auxin, abscisic acid, jasmonic acid and ethylene), and external biotic (pathogen) and abiotic (light conditions, cold and salt stresses) cues (Sun 2011, Current Biology 21, R338-R345). Drought is one of the most important environmental constraints limiting plant growth and agricultural productivity. Unsurprisingly, there is a positive correlation between improved drought tolerance with a more extensive root system including deeper roots and more lateral roots both of which enable soil exploration and below-ground resources acquisition (Yu et al 2008, Plant Cell 20, 1134-1151; Werner et al 2010, Plant Cell 22, 3905-3920). Thus it follows that a common agricultural target is the optimization of root system architecture in order to help overcome yield limitations in crop plants caused by water or nutrient shortages. However, of all the abiotic stresses that curtail crop productivity, drought is the most devastating one and the most recalcitrant to breeder's efforts. Classic breeding approaches are difficult because the trait is governed by many genes and is difficult to score (Werner et al 2010, Plant Cell 22, 3905-3920). While marker-assisted selection (MAS), quantitative trait loci (QTL) and other genomic approaches are being widely used to assist breeding efforts to produce drought-resilient cultivars (Tuberosa and Salvi, 2006, Trends in Plant Science, 11:405-412) the system is limited to the variation present in the screening population.

Interestingly, rice has only one DELLA protein (SLR1), Maize has two (d8 and d9)(Lawit et al 2010, Plant Cell Physiol 51, 1854-1868) while Arabidopsis has five (GA1, RGA, RGL1, RGL2 and RGL3) (Achard and Genschik 2009, J. Exp. Bot. 60, 1085-1092). Furthermore, in a recent phylogenetic analysis Chen et al 2013 found five out of the six grass species they analysed had only a single DELLA while 14 out of the 18 dicot species had two or more DELLA proteins. In contrast, there are 6 members of the BZR family in rice, 10 in maize (www<dot>Grassius<dot>org) and 6 in Arabidopsis (Wang et al 2002, Developmental Cell 2, 505-513).

The growth and development of plants relies on numerous connections between signalling pathways that provides the high developmental plasticity demanded by their sessile life habit (Gallego-Bartolome et al 2012, PNAS 109, 13446-13451). Thus rather than each hormone-signalling pathway existing as an insulated module current evidence indicates that there is a high degree of interaction between different pathways and that a given hormone frequently modulates the output triggered by the rest. By example, it has recently been shown that the cross talk between the GA and BR signalling pathways involves direct interaction between DELLAs and BZR1/BES1 whereby DELLA proteins not only affect the protein stability but also inhibit the transcriptional activity of BZR1 (Li and He 2013, Plant Signaling and Behaviour 8:7, e24686 and references therein). Thus the promotion of cell elongation by GA is partly through the removal of the DELLA-mediated inhibition of BZR1.

It has recently been demonstrated that plant growth and development can be modified through direct manipulation of the master growth regulators DELLA (Lawit, Kundu, Rao and Tomes, 2007, ISOLATED POLYNUCLEOTIDE MOLECULES CORRESPONDING TO MUTANT AND WILD-TYPE ALLELES OF THE MAIZE D9 GENE AND METHODS OF USE, WO 2007124312 A2) and BZR1 (Chory and Wang, 2005, GENES INVOLVED IN BRASSINOSTEROID HORMONE ACTION IN PLANTS, U.S. Pat. No. 6,921,848 B2).

Steroid hormones play an essential role in the coordination of a wide range of developmental and physiological processes in both plants and animals (Thummel and Chory 2002, Genes Dev. 16, 3113-3129). In plants the steroid hormone brassinosteroid (BR) has extensive effects on growth, development and responses to both biotic and abiotic stresses (Zhu et al 2013, Development 140, 1615-1620; Clouse 2011, Plant Cell 23, 1219-1230). In contrast to animal steroid hormone signalling, which functions through nuclear receptors, in plants BRs bind to the extracellular domain of the cell surface receptor kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1) and activate an intracellular signal transduction cascade that regulates gene expression (Clouse 2011, Plant Cell 23, 1219-1230; Kinoshita et al 2005, Nature 433, 167-171). There are multiple steps involving activation and inactivation of intermediates leading to the phosphorylation of two transcription factors, BRASSINAZOLE RESISTANT 1 (BZR1) and BZR2 (also known as BES1). Thus the signal transduction BZR transcription factors are the target components converting signalling into BR responsive gene expression.

There is an emerging pattern in plant hormone signalling where the target transcription factors activated by hormones are also negatively regulated by specific repressor complexes. For example, in the jasmonic acid (JA), auxin, abscisic acid (ABA) and strigolactone (SL) signalling pathways the target transcription factors are negatively regulated by repressor complexes utilising TOPLESS (TPL) as a common co-repressor recruited by a hormone pathway specific repressor (Pauwels et al 2010, Nature 464, 788-791). In the JA transduction pathway the JASMONATE ZIM DOMAIN (JAZ) family of transcriptional repressors both interact with the target JA-responsive transcriptional activator MYC2 and recruit TPL, either directly or via the adaptor protein NOVEL INTERACTOR OF JAZ (NINJA) (Pauwels et al 2010 Nature 464, 788-791).

Accordingly, the ability to regulate the GA and BR pathways to influence many different agricultural traits of interest is of considerable value to commercial agriculture.

The Applicant's Invention

As discussed above, the present invention relates to a method for increasing root biomass in plants by ectopic expression of PEAPOD.

Without wishing to be bound by theory, the applicants have shown that PEAPOD (PPD) appears to be involved in the modulation of both the GA and BR pathways either through directly or indirect interaction with the master growth regulators DELLA and BZR.

Analysis of the primary amino add structure of PPD proteins indicates the presence of a highly conserved novel plant specific domain present only these proteins. There are homologues of PPD in a wide range of eudicot, conifers and some monocot plants (palms, banana, orchids, duckweed) but not Poaceae (grasses).

The PPD genes of Arabidopsis encode proteins that are members of the plant-specific TIFY family, named after the core TIF[F/Y]XG (SEQ ID NO: 146) motif found within a domain known as ZIM (Vanholme et al 2007 Trends Plant Sci. 12, 239-244). The two Arabidopsis PPD proteins, PPD1 and PPD2, are included in the same class II TIFY group as twelve well characterised JAZ proteins that act as repressors of jasmonate responses. However, the PPD proteins and the one other non-JAZ protein in the group do not appear to be involved in responses to jasmonate hormone signalling (Pauwels et al 2010 Nature 464, 788-791).

Again, without wishing to be bound by theory, the applicants propose that the increases in root biomass, according to the invention, are mediated by a new mechanism for regulating both the GA and BR pathways using the PPD gene. Examples 3 and 4 below support this proposal,

This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be better understood with reference to the accompanying drawings in which are described as follows:

FIG. 1 shows the 46 amino acid residues comprising the PEAPOD region from a range of plant species, identical residues are shown by an asterisk.

FIG. 2 shows the internal 27 amino acid residues within the PEAPOD region from a range of plant species, identical residues are shown by an asterisk.

FIG. 3 shows the 6 amino acid residues of the TIFY domain on PEAPOD proteins from a range of plant species, identical residues are shown by an asterisk.

FIG. 4 shows a schematic representation of the PPD protein and the approximate location of conserved PPD, TIFY and Jas* regions

FIG. 5 shows the dimerization of PPD and the interaction between TPL and NINJA in Y2H assays.

FIG. 6 shows the interaction between PPD and NINJA and the interaction between TPL and BZR1 in Y2H assays.

FIG. 7 shows the interaction between PPD, NINJA, TPL and BZR1 in young (A and B) and old (C) leaves using BiFC assays.

FIG. 8 shows a schematic representation of the PPD-NINJA-TPL-BZR1 complex.

FIG. 9 shows the interaction between PPD and BZR1 in Y2H assays.

FIG. 10 shows the response of Wild Type, Δppd mutant, and PEAPOD overexpressor (PPD-OX) hypocotyl length to exogenous GA and PAC applications.

FIG. 11 shows the increase in root growth of ryegrass plants over expressing PPD (PPD-OX) from Arabidopsis thaliana or PEAPOD from Abroella trichopoda compared to the wild type and vector control.

FIG. 12 shows the increase in root length of alfalfa plants over expressing PPD (PPD-OX) compared to the control plants.

FIG. 13 shows that the PEAPOD proteins from Arabidopsis thaliana; Picea sitchensis, Amborella trichopoda, Musa acuminate, Trifolium repens and Selaginella moellendorffii are functionally equivalent. An optimized PEAPOD coding sequence from each was used to complement the PEAPOD deletion mutant Δppd Arabidopsis thaliana (ecotype Landsberg erecta). Seedling images were taken at an equivalent developmental stage.

EXAMPLES

The invention will now be illustrated with reference to the following non-limiting example.

Example 1 Characterisation of PEAPOD Genes Multiple Plant Species

To identify PPD gene orthologues in other plant species the conserved PPD region (46 amino acids) from the Arabidopsis PPD1 gene (SEQ ID NO: 27) was used for searches of public plant gene sequence databases using the search programmes TBLASTN and BLASTP (Altschul et al 1990). PEAPOD sequences were identified from a diverse range of plant species including the mosses, conifers, all orders of dicotyledonous examined and some of the monocotyledonous orders, including: palms, bananas, orchids and duckweed. PEAPOD sequences are not found in the grasses. Representative PEAPOD protein and nucleic acid sequences are shown in SEQ ID NO: 1-26 and SEQ ID NO:83-107 respectively.

The 46 amino acid PEAPOD region from Arabidopsis thaliana PPD1 is shown in SEQ ID NO:27. This region from polypeptides SEQ ID NO: 1-26 was aligned by vector NTI (VNTI) as shown in FIG. 1.

SEQ ID NO:28 shows the consensus for this 46 amino acid PPD region. SEQ ID NO:29 shows the same consensus region but shows which amino acids can be present at each of the variable positions.

A 27 amino acid subsequence from within the 46 amino acid PEAPOD region from Arabidopsis thaliana PPD1 is shown in SEQ ID NO:30.

Alignment of this 27 amino acid subsequence for reach of the same sequences as in FIG. 1, is shown in FIG. 2.

SEQ ID NO:30 shows the consensus for this 27 amino acid PPD region. SEQ ID NO:32 shows the same consensus region but shows which amino acids can be present at each of the variable positions.

In each of the PPD peptide sequences of SEQ ID NO: 1-26 there is also a conserved TIFY motif which is located after the 46 amino acid PPD region. The number of amino acid residues separating the C-terminus of the PPD region and the N-terminus of the TIFY motif depends on the source of the PPD; for example the number varies between 46 to 140 amino acids for SEQ ID NO:1-26.

SEQ ID NO: 33 shows the Arabidopsis PPD1 sequence over the TIFY motif. The alignment of the TIFY motif (as described by Vanholme et al 2007 Trends Plant Sci. 12, 239-244) from SEQ ID NO:1-26 is shown in FIG. 3.

SEQ ID NO:34 shows the consensus for this 6 amino acid TIFY motif. SEQ ID NO:35 shows the same consensus region but shows which amino acids can be present at each of the variable positions.

Completely conserved residues in the PPD and TIFY domains are highlighted with asterisks in FIGS. 1-3.

The applicants assert that these regions and motifs described above are found in all PEAPOD proteins identified and are diagnostic for such PEAPOD proteins

Example 2 Demonstrating PEAPOD Functionality of PEAPOD Sequences from Multiple Plant Species

The functionality of any PEAPOD sequence can be confirmed by complementation of the Arabidopsis Δppd mutant leaf phenotype. Complementation of the Arabidopsis Δppd mutant leaf phenotype was first used to identify the Arabidopsis PPD gene (White 2006). This was seen by a restoration of the wild type flattened leaf phenotype and normal rosette shape as opposed to the domed leaf and the twisting of the rosette to a “propeller” phenotype.

PEAPOD sequences, such as those of SEQ IN NO: 1-26 (including: palm, conifer, moss, orchid and other dicot species) or any other PEAPOD sequence to be tested can be transformed into the Arabidopsis Δppd mutant by methods well known to those skilled in the art. An example of such a method is described below.

Cloning and Gene Constructs

Generation of CaMV35s:: Arabidopsis thaliana PPD1 Construct for over Expression of Arabidopsis PPD1 in the Arabidopsis Δppd Mutant

An expression construct was synthesised to enable the over expression of Arabidopsis thaliana PPD1 under the CaMV35s promoter (SEQ ID NO.129) in the Arabidopsis Δppd mutant. The PPD ORF was optimised for expression in Arabidopsis; this included a modified Joshi sequence (Joshi 1997, Nucleic Acid Research 15, 6643-6653), optimisation of condons, removal of mRNA instability sequences, removal of polyA signal sequences, removal of cryptic splice sites, addition of a BamHI removable C-terminal V5 epitope and His tag tail (encoding SEQ ID NO:37) and addition of a double stop codon. The construct (with and without the tail) was then placed between the CaMV35s promoter and ocs terminator by the GATEWAY® LR reaction, which coded for SEQ ID NO:105 and SEQ ID NO:111 respectively.

Generation of CaMV35s:: Trifolium repens PPD Construct for over Expression of Trifolium repens PPD1 in the Arabidopsis Δppd Mutant

An expression construct was synthesised to enable the over expression of Trifolium repens PPD under the CaMV35s promoter (SEQ ID NO. 129) in the Arabidopsis Δppd mutant. The PPD ORF was optimised for expression in Arabidopsis; this included a modified Joshi sequence (Joshi 1997, Nucleic Acid Research 15, 6643-6653), optimisation of condons, removal of mRNA instability sequences, removal of polyA signal sequences, removal of cryptic splice sites, addition of a BamHI removable C-terminal V5 epitope and His tag tail (encoding SEQ ID NO:37) and addition of a double stop codon. The construct (with and without the tail) was then placed between the CaMV35s promoter and ocs terminator by the GATEWAY® LR reaction, which coded for SEQ ID NO:106 and SEQ ID NO:112 respectively.

Generation of CaMV35s:: Amborella trichopoda PPD Construct for over Expression of Amborella trichopoda PPD in the Arabidopsis Δppd Mutant

An expression construct was synthesised to enable the over expression of Amborella trichopoda PPD under the CaMV35s promoter (SEQ ID NO. 129) in the Arabidopsis Δppd mutant. The PPD ORF was optimised for expression in Arabidopsis; this included a modified Joshi sequence (Joshi 1997, Nucleic Acid Research 15, 6643-6653), optimisation of condons, removal of mRNA instability sequences, removal of polyA signal sequences, removal of cryptic splice sites, addition of a BamHI removable C-terminal V5 epitope and His tag tail (encoding SEQ ID NO:37) and addition of a double stop codon. The construct (with and without the tail) was then placed between the CaMV35s promoter and ocs terminator by the GATEWAY® LR reaction, which coded for SEQ ID NO:107 and SEQ ID NO:113 respectively.

Generation of CaMV35s:: Musa acuminate PPD Construct for over Expression of Musa acuminate PPD in the Arabidopsis Δppd Mutant

An expression construct was synthesised to enable the over expression of Musa acuminate PPD under the CaMV35s promoter (SEQ ID NO. 129) in the Arabidopsis Δppd mutant. The PPD ORF was optimised for expression in Arabidopsis; this included a modified Joshi sequence (Joshi 1997, Nucleic Acid Research 15, 6643-6653), optimisation of condons, removal of mRNA instability sequences, removal of polyA signal sequences, removal of cryptic splice sites, addition of a BamHI removable C-terminal V5 epitope and His tag tail (encoding SEQ ID NO:37) and addition of a double stop codon. The construct (with and without the tail) was then placed between the CaMV35s promoter and ocs terminator by the GATEWAY® LR reaction, which coded for SEQ ID NO:108 and SEQ ID NO:114 respectively.

Generation of CaMV35s:: Picea sitchensis PPD1 Construct for over Expression of Picea sitchensis PPD in the Arabidopsis Δppd Mutant

An expression construct was synthesised to enable the over expression of Picea sitchensis PPD under the CaMV35s promoter (SEQ ID NO. 129) in the Arabidopsis Δppd mutant. The PPD ORF was optimised for expression in Arabidopsis; this included a modified Joshi sequence (Joshi 1997, Nucleic Acid Research 15, 6643-6653), optimisation of condons, removal of mRNA instability sequences, removal of polyA signal sequences, removal of cryptic splice sites, addition of a BamHI removable C-terminal V5 epitope and His tag tail (encoding SEQ ID NO:37) and addition of a double stop codon. The construct (with and without the tail) was then placed between the CaMV35s promoter and ocs terminator by the GATEWAY® LR reaction, which coded for SEQ ID NO:109 and SEQ ID NO:115 respectively.

Generation of CaMV35s:: Selaginella moellendorffii PPD1 Construct for over Expression of Selaginella moellendorffii PPD in the Arabidopsis Δppd Mutant

An expression construct was synthesised to enable the over expression of Selaginella moellendorffii PPD under the CaMV35s promoter (SEQ ID NO. 129) in the Arabidopsis Δppd mutant. The PPD ORF was optimised for expression in Arabidopsis; this included a modified Joshi sequence (Joshi 1997, Nucleic Acid Research 15, 6643-6653), optimisation of condons, removal of mRNA instability sequences, removal of polyA signal sequences, removal of cryptic splice sites, addition of a BamHI removable C-terminal V5 epitope and His tag tail (encoding SEQ ID NO:37) and addition of a double stop codon. The construct (with and without the tail) was then placed between the CaMV35s promoter and ocs terminator by the GATEWAY® LR reaction, which coded for SEQ ID NO:110 and SEQ ID NO:116 respectively.

Plant Materials and Growth Conditions

Arabidopsis thaliana (L.)Heynh ecotype Ler can be used as wild-type (WT). The Δppd loss of function deletion mutant (with PPD1 and PPD2 deleted) is as previously described in White 2006 PNAS 103, 13238-13243.

Plants are grown in a temperature-controlled glasshouse at a continuous 21° C. or in a controlled environment cabinet at 23° C. in 16-h light_8-h dark cycles.

Transformation of Arabidopsis

Constructs above can be transformed into Arabidopsis by the floral dip infiltration method (Clough and Bent, 1998 Plant J 16, 735-43). The Δppd line is transformed to express the PPD polypeptides by standard techniques. Transgenic plants are confirmed by standard PCR analysis techniques with a combination of transgene-specific and T-DNA primers.

Complementation of the Δppd line to produce a wild-type leaf and rosette phenotype in T1 seedlings (the off-spring of the infiltrated plant) confirms PEAPOD functionality of the introduced gene, which can be shown in photographs.

This approach can be use to confirm the PEAPOD functionality of any gene which the applicant asserts, demonstrates it suitability of use in the present invention.

The PEAPOD proteins from Arabidopsis thaliana; Picea sitchensis, Amborella trichopoda, Musa acuminate, and Selaginella moellendorffii were shown to be functionally equivalent by the complementation of the PEAPOD deletion mutant Δppd Arabidopsis thaliana ecotype Landsberg erecta (FIG. 13).

Example 3 PEAPOD may be Involved in Regulating the Brassinosteroid Signalling Pathway

The applicants used yeast two hybrid (Y2H) assays Bi-molecular fluorescence (BiFC) to investigate the interactions between PPD, NINJA, TPL and BZR1.

Cloning and Constructs

The constructs for Y2H and BiFC assays were generated as follows. Arabidopsis DNA sequences encoding the open reading frames for; At4g14713 (PPD1) and truncation and deletion derivatives of PPD1: PPD1; PPD1Δppd (N-terminal truncation of sequences encoding aa 1-61), PPD1Δtify, (internal deletion of sequences encoding aa 154-186), PPD1Δjas*(C-terminal truncation of sequences encoding aa 229-313) (FIG. 4), At4g28910 (NINJA), At1g15750 (TPL), At1g75080 (BZR1), a synthetic PUAS-35S promoter, and sequences encoding GAL4DBD and c-myc fusion proteins were synthesised and sequence verified by GeneArt. Most sequences were supplied as clones in pENTR221 ready for Gateway cloning into yeast and plant expression vectors. The exception, a promoter sequence for in planta transcription activation assays, incorporating 5′ Xho1 and 3′ Nco1 restriction enzyme sites, was supplied cloned in pMA-RQ. Plasmids for the transient LUC reporter assay: A synthetic promoter with 5×UAS GAL4 DNA binding sites upstream of a −105 bp CaMV35S promoter was cloned into the XhoI-NcoI sites within a dual luciferase construct pNWA62, which contains an intron-containing Firefly Luciferase gene(LUC) and 35Spro::Renilla Luciferase (REN) as an internal standard, to construct pAML7. For the over expression of GAL4DBD fusion proteins DNA sequences encoding a GAL4 DNA-binding domain (GAL4DBD aa 1-147) and N-terminal GAL4DBD fusions (using a linker encoding GGGGS) with 2× the VP16 activator domain (GAL4DBD-VP16) or PPD1 (GAL4DBD-PPD1), were cloned using Gateway technology into pRSh1 (Winichayakul et al 2008) to construct vectors pRSh1-GAL4DBD, pRSh1-GAL4DBD-VP16, and pRSh1-GAL4DBD-PPD1 for expression of the fusion proteins in planta.

Plasmids for Yeast Two-hybrid Analysis

Full length coding sequences of BZR1, NINJA, TPL, and PPD1, together with truncation or deletion derivatives of PPD1 (PPD1Δppd, PPD1Δtify, and PPD1.4jas*), were Gateway sub-cloned into pDEST32 (N-terminal GAL4DBD) or pDEST22 (N-terminal GAL4AD), to construct pDEST32-PPD1, pDEST32-PPD1Δppd, pDEST32-PPD1Δtify, pDEST32-PPD1.4jas*, pDEST32-TPL, as bait vectors and pDEST22-PPD1, pDEST22-BZR1, and pDEST22-NINJA as prey vectors. When expressed these constructs produced proteins listed in SEQ ID NO: 56-67, and 70-72; including: DNA binding domain (DBD), activation domain (AD), PPD1 fused to DBD (PPD1-DBD), PPD1 fused to AD (PPD1-AD), PPD1 with no TIFY domain fused to AD (PPD1-tify-AD), PPD1 with no jas domain fused to AD (PPD1-jas*-AD), TOPLESS (TPL), TPL fused to DBD (TPL-DBD), NINJA, NINJA fused to AD (NINJA-AD), BZR1 fused to AD (BZR1-AD), PPD1 minus the ppd domain fused to DBD (PPD1-Δppd-DBD), PPD1 minus the TIFY domain fused to DBD (PPD1-tify-DBD), PPD1 minus the jas domain fused to DBD (PPD1-jas*-DBD).

Plasmids for Bimolecular Fluorescence Complementation

The binary BiFC-Gateway YFP vectors pDEST-VYNE(R)^(GW) (Venus aa 1-173) and pDEST-VYCE(R)^(GW) (Venus aa 156-239) with N-terminal fusions, were used to construct the following vectors; pDESTnYFP-BZR1, pDESTnYFP-NINJA, pDESTnYFP-PPD1, pDESTcYFP-BZR1, pDESTcYFP-PPD1, pDESTcYFP-PPD1Δppd, pDESTcYFP-PPD1Δtify and pDESTcYFP-PPD1Δjas*. For transient in planta expression of proteins interacting with PPD1 or BZR, NINJA and TPL were Gateway® sub-cloned into pRSh1, to construct pRSh1-NINJA and pRSh1-TPL. Plasmids for co-immunoprecipitation: A synthesised DNA construct encoding PPD1 with a 3×c-myc C-terminal fusion was sub-cloned into pRSh1 to produce pRSh1-PPD1-3×c-myc, while the NINJA cDNA sequence was sub-cloned into pB7FWG2,0 (Karimi et al 2002, Trends Plant Sci. 7, 193-195) to construct pB7FWG2-NINJA-GFP. When expressed these constructs produced proteins listed in SEQ ID NO: 63, 65, 73-82: including TOPLESS (TPL), NINJA, Bimolecular Fluorescence (BiFC) nYFP, BiFC cYFP, BiFC nYFP-NINJA, BiFC nYFP-BZR1, BiFC cYFP-PPD1, BiFC cYFP-NINJA, BiFC cYFP-BZR1, BiFC cYFP-PPD1-ppd, BiFC cYFP-PPD1-tify, BiFC cYFP-PPD1-jas*.

The ProQuest two-hybrid system (Invitrogen) was used to analyse interactions between PPD1, NINJA, TPL, and BZR1. Combinations of bait and prey constructs were used to co-transform yeast strain MaV203 (Invitrogen), with selection on synthetic dropout (SD) SD/-Leu/-Trp agar plates. Transformed strains were tested for interactions using 10 μl droplets of 1 in 10 and 1 in 100 dilutions on SD/-Leu/-Trp/-His plates with different concentrations of 3-aminotriazol (3-AT) (Sigma).

Transient BiFC experiments were performed using combinations of pDESTnYFP and pDESTcYFP plasmids, with or without plasmids for the expression of NINJA (pRSh1-NINJA) or TPL (pRSh1-TPL) and Agrobacterium-infiltration of Nicotiana benthamiana leaves. For infiltration Agrobacterium tumefaciens GV3101 strains containing the binary vectors were re-suspended from plates and prepared for transformation as described for the LUC assay. All YFP and expression strains were mixed in ratios of 1:1 (vol/vol) with the addition of strain P19 at 1/10th volume. Five leaf discs were sampled from each infiltrated leaf after 40 h. Two hours prior to sampling for microscopic fluorescence observations leaves were infiltrated with a 1 μg/ml DAPI solution to stain nuclei. YFP fluorescence and DAPI staining was detected using an Olympus Fluoview FV10i confocal laser scanning microscope. Each experiment was repeated twice.

Y2H screening using PPD1 as a bait protein identified NINJA as a direct interactor with PPD1. Results from BiFC assays suggested PPD1 interacted with NINJA in plants, and that the TIFY motif was also essential for this interaction (FIG. 7). It is possible that NINJA functions as a bridge between TPL and PPD1. Using Y2H no direct interaction between PPD1 and BZR1 was observed (FIG. 5). However, recent tandem affinity purification (TAP) experiments have shown that TPL may interact with BZR1 (Wang et al 2013, Mol. Cell. Proteomics 12, 3653-3665), and here Y2H results confirmed that a direct interaction occurs (FIG. 6).

To determine the molecular function of the PPD proteins the interactions of PPD1, NINJA, TPL, and BZR1 were studied in planta. Bimolecular fluorescence (BiFC) was used to show that in the pavement cells of immature Nicotiana benthamiana leaves PPD1 appears to interact with BZR1 in the nucleus (FIG. 7A,B). The NINJA-binding TIFY motif in PPD1 was essential for this interaction. Moreover, no interaction was observed when nYFP-PPD1 and cYFP-BZR1 were co-expressed in fully expanded leaves (FIG. 7C). Interestingly, interaction between PPD1 and BZR1 was restored upon co-expression of NINJA but not TPL alone, suggesting the lack of interaction in the mature leaf was due to a limitation of endogenous NINJA. As for immature leaves, interaction between PPD1 and BZR1, even in the presence of NINJA and TPL co-expression, was not observed when the PPD1 NINJA-binding TIFY motif was deleted (FIG. 7C). These results suggest that PPD1, NINJA, TPL and BZR1 exist as a complex in plants and that NINJA is required to recruit PPD1 to interact via TPL with BZR1.

PPD1 does not appear to directly interact with the target BZR1 transcription factor. Instead the results of PPD1 protein interaction experiments suggest a model in which the PPD proteins recruit TPL transcriptional co-repressors, using NINJA as an adaptor, and this PPD-NINJA-TPL complex interacts with the EAR motif of the BZR transcription factors (FIG. 8). Thus in this model the PEAPOD1 (PPD1) protein of Arabidopsis thaliana would act as a repressor of the BR signalling pathway and in combination with NINJA and TPL, negatively regulates BZR1.

Example 4 PEAPOD may be Involved in Regulating the Gibberellin Signalling Pathway

Giberellic acid (GA) treatment is known to reduce levels of the DELLA proteins (including RGA1) which are GA repressors; to determine the relationship between PPD, DELLA and the GA signalling pathway the applicants performed a yeast two-hybrid (Y2H) analysis between PPD and DELLA (RGA1) and applied gibberellic acid (GA) hormone and GA biosynthesis inhibitor (paclobutrazol, PAC) to wild type, Δppd mutant, and the Δppd mutant PPD over expressor (PPD-OX).

The ProQuest two-hybrid system (Invitrogen) was used to analyse interactions between PPD1, and RGA1. Full length coding sequences of PPD1, together with truncation or deletion derivatives of PPD1 (PPD1Δppd, PPD1Δtify, and PPD1Δjas*) (FIG. 4), were Gateway sub-cloned into pDEST32 (N-terminal GAL4DBD) or pDEST22 (N-terminal GAL4AD). When translated these generated the following peptide sequences od SEQ ID NO: 58, 68, 69, 70, 71, 72, which are PPD1-DBD, RGA1, RGA1-AD, PPD1-ppd-DBD, PPD1-tify-DBD, PP1-jas*-DBD respectively.

Combinations of bait and prey constructs were used to co-transform yeast strain MaV203 (Invitrogen), with selection on synthetic dropout (SD) SD/-Leu/-Trp agar plates. Transformed strains were tested for interactions using 10 μl droplets of 1 in 10 and 1 in 100 dilutions on SD/-Leu/-Trp/-His plates with different concentrations of 3-aminotriazol (3-AT) (Sigma). The PPD1-RGA1 interaction was tested with PPD1-DBD used as bait. Transformed yeast was spotted as a ten-fold dilution on control medium (−2) or selective medium (−3) with 15 mM 3AT. Controls were empty vectors, DBD, GAL4 DNA binding domain, AD, GAL4 activation domain (FIG. 4). The Y2H results suggests that PPD can directly bind to DELLA (FIG. 9).

For exogenous applications of GA or PAC seeds were surface sterilised with 70% ethanol, 0.01% Triton X-100 for 10 min, followed by 100% ethanol for 5 min, air dried on sterile filter paper, and transferred to media plates containing half-strength MS salts, 1% sucrose and 0.8% agar. Plates were incubated for 5 days at 4° C. in the dark then transferred to 24° C. with a 14 h light/10 h dark daily cycle. Light was provided by fluorescent tubes (Philips TLD 58W/865) at an intensity of 100 μM m⁻² s⁻¹. Wild-type (Col-0) Δppd mutant and transgenic PPD-OX seedlings were grown for five days on medium with different concentrations of GA (FIG. 10A) or PAC (FIG. 10B). GA (ACROS organics), and PAC (Sigma-Aldrich) were dissolved in ethanol and acetone respectively, filter sterilised and incorporated into media plates. Ethanol or acetone (0.5%) was used for mock treatments. Seedlings were grown at 24° C. under a 14 h light/10 h dark daily cycle for 5 days before hypocotyl lengths were analysed (n=35). Each treatment was repeated twice; error bars=standard error of the mean.

A reduction of DELLA leads to an increase in transcription of DELLA target genes promoting cell expansion and can be quantified by measuring hypocotyl elongation of seedlings growing on media containing varying levels of GA. The lowest concentration of GA (1 μM) did not promote elongation of the wild type (WT) hypocotyl whereas both the loss-of-function PPD mutant (Δppd) and the transgenic PPD over expressing (PPD-OX) seedlings showed increased hypocotyl elongation (FIG. 10A). At higher GA concentrations (5-50 μM) elongation of the WT hypocotyl occurred in a dose dependent manner. In comparison the Δppd and PPD-OX seedlings showed hypersensitive elongation up to 5 and 10 μM GA respectively where they both reached approximately the same length (FIG. 10A).

GA biosynthesis is inhibited by applications of exogenous paclobutrazol (PAC); this results in an increase in the DELLA repressor proteins and corresponding reduction in cell expansion. Wild type seedlings demonstrated a dose dependent decrease of hypocotyl elongation from 0 to 10 μM PAC (FIG. 10B). Once again the Δppd seedlings demonstrated a hyper sensitive response which was seen as a larger reduction in hypocotyl elongation over the same range of PAC applications. The PPD-OX seedlings however, were relatively insensitive until the PAC concentration was increased beyond 0.1 μM, after which they too showed a decrease in hypocotyl length (FIG. 10B).

The hypersensitive response to GA by the Δppd seedlings potentially reflects the combination of increased targeting of DELLA for degradation in the absence of transcription factor repression by PPD. Similarly, the addition of PAC in the Δppd background possibly leads to a greater reduction in hypocotyl elongation compared to WT because it is done in the absence of one of DELLAs natural antagonists—PPD, suggesting PPD and GA compete for binding to DELLA.

It can be predicted that the over expression of PPD would result in a higher level of antagonism of DELLA, as such the hypocotyl elongation of these plants ought to be hypersensitive to GA; indeed this is what we observed in the PPD-OX seedlings. In the reverse situation when the GA level was reduced (by the application of PAC) the PPD-OX seedlings were unresponsive until the PAC concentration was greater than 0.1 μM. This likely reflects the point at which there was a sufficient reduction in endogenous GA levels to see the influence of DELLA protein not antagonised by the over expressed PPD.

Example 5 Increasing Root Biomass by Increasing Expression of PEAPOD in Plants

Constructs

Described below are several constructs for expressing PEAPOD sequences from various species, under the control of various promoters, for expression in dicotyledonous and monocotyledonous plants.

Generation of pTobRB7 Δ1.3::PPD Dicot Construct for (Root-preferred) over-expression of Arabidopsis PPD in Dicotyledonous Plants

Construs were synthesised containing the coding sequence of PPD1 for expression in roots of dicots, the nucleic acid sequences are shown in SEQ ID NO:40 and 126. The PPD ORF was originally from the Arabidopsis thaliana PPD1 cDNA (Accession No. NM_202819) and was modified to include a modified Joshi sequence (Joshi 1997, Nucleic Acid Research 15, 6643-6653), removal of cryptic splice sites, in the case of SEQ ID NO: 40 the inclusion of the third intron from Arabidopsis thaliana DGAT1 (SEQ ID NO:39), addition of a BamHI removable C-terminal V5 epitope and His tag tail (encoding SEQ ID NO:37) and addition of a double stop codon. The position of the intron was optimised for splice site prediction, performed by NetGene2 (www<dot>cbs<dot>dtu<dot>dk/services/NetGene2/).

The construct of SEQ ID NO: 40 (with and without the tail) was then placed between the TobRB7 Δ1.3 promoter (Yamamoto et al 1991, Plant Cell, 3:371-382) and NOS terminator by the GATEWAY® LR reaction, to create SEQ ID NO:42 and SEQ ID NO:44 which coded for SEQ ID NO:36 and SEQ ID NO:38 respectively. A similar subcloning step was performed on the construct shown in SEQ ID NO: 126 (with and without the C-terminal tail), which coded for SEQ ID NO: 108 and SEQ ID NO: 114 respectively.

Generation of pTobRB7 Δ0.6::PPD Dicot Construct for (Root-preferred) over-expression of Arabidopsis PPD in Dicotyledonous Plants

Constructs were synthesised containing the coding sequence of PPD1 for expression in roots of dicots, the nucleic acid sequences are shown in SEQ ID NO:40 and 126. The PPD ORF was originally from the Arabidopsis thaliana PPD1 cDNA (Accession No. NM_202819) and was modified to include a modified Joshi sequence (Joshi 1997, Nucleic Acid Research 15, 6643-6653), removal of cryptic splice sites, in the case of SEQ ID NO: 40 the inclusion of the third intron from Arabidopsis thaliana DGAT1 (SEQ ID NO:39), addition of a BamHI removable C-terminal V5 epitope and His tag tail (encoding SEQ ID NO:37) and addition of a double stop codon. The position of the intron was optimised for splice site prediction, performed by NetGene2 (www<dot>cbs<dot>dtu<dot>dk/services/NetGene2/).

The construct (with and without the tail) was then placed between the TobRB7 Δ0.6 promoter (Yamamoto et al 1991, Plant Cell, 3:371-382) and NOS terminator by the GATEWAY® LR reaction, to create SEQ ID NO:43 and SEQ ID NO:45 which coded for SEQ ID NO:36 and SEQ ID NO:38 respectively. A similar subcloning step was performed on the construct shown in SEQ ID NO: 126 (with and without the C-terminal tail), which coded for SEQ ID NO: 108 and SEQ ID NO: 114 respectively.

Generation of pAtWRKY6::PPD Dicot Construct for (Root-preferred) over-expression of Arabidopsis PPD in Dicotyledonous Plants

Constructs were synthesised containing the coding sequence of PPD1 for expression in roots of dicots, the nucleic acid sequence is shown in SEQ ID NO:41 and 126. The PPD ORF was originally from the Arabidopsis thaliana PPD1 cDNA (Accession No. NM_202819) and was modified to include a modified Joshi sequence (Joshi 1997, Nucleic Acid Research 15, 6643-6653), removal of cryptic splice sites, in the case of SEQ ID NO: 41 the inclusion of the third intron from Arabidopsis thaliana DGAT1 (SEQ ID NO:39), addition of a BamHI removable C-terminal V5 epitope and His tag tail (encoding SEQ ID NO:37) and addition of a double stop codon. The position of the intron was optimised for splice site prediction, performed by NetGene2 (www<dot>cbs<dot>dtu<dot>dk/services/NetGene2/).

The construct (with and without the tail) was then placed between the tobacco AtWRKY6 promoter (Robatzek and Somssich 2001, The Plant Journal, 28: 123-133) and NOS terminator by the GATEWAY® LR reaction, to create SEQ ID NO:44 and SEQ ID NO:47 which coded for SEQ ID NO:36 and SEQ ID NO:38 respectively. A similar subcloning step was performed on the construct shown in SEQ ID NO: 126 (with and without the C-terminal tail), which coded for SEQ ID NO: 108 and SEQ ID NO: 114 respectively.

Generation of pTobRB7 Δ1.3::PPD Monocot Construct for (Root-preferred) over-expression of Arabidopsis PPD in Monocotyledonous Plants

Construct were synthesised containing the coding sequence of PPD1 for expression in roots of monocots, the nucleic acid sequences are shown in SEQ ID NO:49 and SEQ ID NO: 120. The PPD ORF was optimised for expression in monocotyledonous plants; this included a modified Joshi sequence (Joshi 1997, Nucleic Acid Research 15, 6643-6653), optimisation of codons, removal of mRNA instability sequences, removal of polyA signal sequences, removal of cryptic splice sites, in the case of SEQ ID NO: 49 the inclusion of the third intron from Lolium perenne DGAT1 (SEQ ID NO:48), addition of a BamHI removable C-terminal V5 epitope and His tag tail (encoding SEQ ID NO:37) and addition of a double stop codon. The position of the intron was optimised for splice site prediction, performed by deepc2 (www<dot>cbs<dot>dtu<dot>dk/services/NetGene2/).

The construct (with and without the tail) was then placed between the pTobRB7 Δ1.3 promoter (Yamamoto et al 1991, Plant Cell, 3:371-382) and NOS terminator by the GATEWAY® LR reaction, to create SEQ ID NO:50 and SEQ ID NO:53 which coded for SEQ ID NO:36 and SEQ ID NO:38 respectively. A similar subcloning step was performed on the construct shown in SEQ ID NO: 120 (with and without the C-terminal tail), which coded for SEQ ID NO: 108 and SEQ ID NO: 114 respectively.

Generation of pTobRB7 Δ0.6::PPD Monocot Construct for (Root-preferred) over-expression of Arabidopsis PPD in Dicotyledonous Plants

Constructs were synthesised containing the coding sequence of PPD1 for expression in roots of monocots, the nucleic acid sequences are shown in SEQ ID NO:49 and SEQ ID NO: 120. The PPD ORF was optimised for expression in monocotyledonous plants; this included a modified Joshi sequence (Joshi 1997, Nucleic Acid Research 15, 6643-6653), optimisation of condons, removal of mRNA instability sequences, removal of polyA signal sequences, removal of cryptic splice sites, in the case of SEQ ID NO: 49 the inclusion of the third intron from Lolium perenne DGAT1 (SEQ ID NO: 48), addition of a BamHI removable C-terminal V5 epitope and His tag tail (encoding SEQ ID NO:37) and addition of a double stop codon. The position of the intron was optimised for splice site prediction, performed by deepc2 (www<dot>cbs<dot>dtu<dot>dk/services/NetGene2/).

The construct (with and without the tail) was then placed between the pTobRB7 Δ0.6 promoter (Yamamoto et al 1991, Plant Cell, 3:371-382) and NOS terminator by the GATEWAY® LR reaction, to create SEQ ID NO:51 and SEQ ID NO:54 which coded for SEQ ID NO:36 and SEQ ID NO:38 respectively. A similar subcloning step was performed on the construct shown in SEQ ID NO: 120 (with and without the C-terminal tail), which coded for SEQ ID NO: 108 and SEQ ID NO: 114 respectively.

Generation of pAtWRKY6::PPD Monocot Construct for (Root-preferred) over-expression of Arabidopsis PPD in Monocotyledonous Plants

Constructs were synthesised containing the coding sequence of PPD1 for expression in roots of monocots, the nucleic acid sequences are shown in SEQ ID NO:49 and SEQ ID NO: 120. The PPD ORF was optimised for expression in monocotyledonous plants; this included a modified Joshi sequence (Joshi 1997, Nucleic Acid Research 15, 6643-6653), optimisation of condons, removal of mRNA instability sequences, removal of polyA signal sequences, removal of cryptic splice sites, in the case of SEQ ID NO: 49 the inclusion of the third intron from Lolium perenne DGAT1 (SEQ ID NO:48), addition of a BamHI removable C-terminal V5 epitope and His tag tail (encoding SEQ ID NO:37) and addition of a double stop codon. The position of the intron was optimised for splice site prediction, performed by deepc2 (www<dot>cbs<dot>dtu<dot>dk/services/NetGene2/).

The construct (with and without the tail) was then placed between the pAtWRKY6 promoter (Robatzek and Somssich 2001 The Plant Journal, 28: 123-133) and NOS terminator by the GATEWAY® LR reaction, to create SEQ ID NO:52 and SEQ ID NO:55 which coded for SEQ ID NO:36 and SEQ ID NO:38 respectively. A similar subcloning step was performed on the construct shown in SEQ ID NO: 120 (with and without the C-terminal tail), which coded for SEQ ID NO: 108 and SEQ ID NO: 114 respectively.

Generation of Expression Constructs Containing PPD from other Plant Species

To demonstrate that PPD from species other than Arabidopsis can be used we synthesized constructs that when placed under a suitable promoter would express PPD from Arabidopsis thaliana (PPD control), the legume Trifolium repens, the primitive angiosperm Amborella trichopoda, the monocotoledonous Musa acuminate, the conifers Picea abies and Picea sitchensis and the lycophyte Selaginella moellendorffii. These were optimized for expression in either rice (including a monocotoledonous intron) or Arabidopsis (no intron). These were engineered to contain a removable tail which considered of two BamHI sites (coding for glycine-serine) on either side of a nucleic acid sequence encoding a V5-His tag. The peptide sequences (with and without the C-terminal V5-His tag tail) are shown in sequences 108-119. The corresponding nucleic acid sequences optimized for expression in rice are shown in sequences 120-125, and optimized for expression in Arabidopsis are shown in sequences 126-131.

The Arabidopsis optimised versions were placed downstream of appropriate promoters in binary vectors by the GATEWAY LR reaction and transformed into Δppd Arabidopsis thaliana by the floral dip method.

The rice optimized versions were placed downstream of appropriate promoters by the GATEWAY LR reaction and transformed into ryegrass biolystically.

Plant Transformation

Transformation of Arabidopsis

Arabidopsis is transformed as described in Example 2.

Transformation of Alfalfa

Alalfa plants over-expressing the Peapod construct were generated by Agrobacterium-mediated transformation using a method adapted from Samac et al. 2000 (Methods in Molecular Biology 343: 301-311).

Plant Tissue

Highly regenerable genotypes of Regen-SY (Bingham 1991 Crop Sci. 31, 1098) were identified with an ability to develop whole plants from somatic embryos under in vitro conditions. A single genotype was selected for use in plant transformation experiments using this protocol.

Agrobacterium Strains and Vectors

The binary vectors pRSh1::PPD-OP and pRSh1::PPD-OP-V5 were used for all transformation experiments. The vector pRSh1 is a derivative of pART27 (Gleave 1992 Plant Mol Biol 20: 1203-1207) and contains the bar selection gene expressed from the CaMV 35S promoter and the grass optimised Peapod gene also under the expression of the CaMV 35S promoter. The plasmid was transferred into A. tumefaciens strain GV310 and transformants were selected on YM plates containing 200 mgl-1 spectinomycin. Successful mobilisation of the plasmid into Agrobacterium was confirmed by restriction mapping following preparation of plasmid DNA from the bacterial culture.

A culture of the binary plasmids was added to 25 mL of Mannitol Glutamate Luria (MGL) broth containing 100 mg/L Spectinomycin. Bacterial cultures were grown overnight (16 hours) on a rotary shaker (200 rpm) at 28° C. Cultures were harvested by centrifugation (3000×g), the supernatant removed and the cells re-suspended in a 5 mL solution of 10 mM MgSO4 in preparation for plant transformation.

Plant Transformation

The second and third fully expanded leaves from a tissue culture responsive genotype of Regen-SY were harvested and rinsed for 30 seconds in a 50% Ethanol solution followed by a wash in sterile distilled water before surface sterilisation in a sodium hypochlorite solution (3% available chlorine). Sterile leaves are then cut into leaf disks and then floated on a solution of SH1.5D callus induction medium. For transformation of the leaf disks, approximately 3 mL Agrobacterium broth to 12 mL SH1.5D and incubated for 15 minutes with occasional stirring to thoroughly coat the leaves with Agrobacterium. The bacterial solution is then removed and the leaf pieces blotted briefly on sterile filter paper before placing adaxial side up on SH1.5D gelled with 0.8% Phytoagar. Plates were then incubated for 5 days in the dark at 25° C.

After inoculation, leaf pieces were removed from plates and rinsed 3-4 times in sterile SH1.5D liquid medium blotted dry on sterile filter paper and placed on SH1.5D plates supplemented with 5 mg/L Ammonium glufosinate and 100 mg/L Cefotaxime and Timentin for selection and incubated for 15 days in the dark at 25° C. Callus developing on leaf pieces were then passaged to fresh SH1.5D selection medium for a further 15 days.

Transformed callus was then transferred to BL0 medium supplemented with Ammonium glufosinate, Cefotaxime and Timentin for formation of somatic embryos. Once embryos developed, they were dissected and transferred to a half strength MSN medium with selection for germination. Whole rooted plants were transferred and established in the glasshouse.

PCR Analysis

PCR analysis was performed to confirm stable integration of the T-DNA into the genome for plants recovered from transformation experiments. Genomic DNA was extracted from approximately 50 mg of in vitro grown leaves using the Genomic DNA Mini Kit (Geneaid).

Primer pairs specific to the ocs3′ polyadenylation signal (ocs3′-1f, 5′-GATATGCGAGACGCCTATGA-3′ [SEQ ID NO: 132]; ocs3′-1r, 5′-GAGTTCCCTTCAGTGAACGT-3′ [SEQ ID NO: 133]) were used to produce an amplification product of 439 bp. Control reactions comprising plasmid DNA template, non-transformed plant DNA or water only were also included. The protocol for PCR reactions consisted of: an initial denaturation of 94° C. for 5 minutes, 30 cycles of 95° C. 30 s, 55° C. 15 s, 72° C. 1 min, and an extension of 72° C. for 10 min. Amplification products were resolved on 1.0% agarose gels by gel electrophoresis in TAE buffer and visualized with a Bio-Rad Gel Doc imaging system.

General Composition of Media used for Alfalfa Transformation

A. MGL Mannitol 5.0 g/L L glutamic acid 1.0 g/L KH2PO4 250 mg/L MgSO4 100 mg/L NaCl 100 mg/L Biotin 100 mg/L Bactotryptone 5.0 g/L Yeast extract 2.5 g/L pH 7.0 (NaOH) B. SH1.5D SH macro and micro salts (Duchefa) SH vitamins (Duchefa) K2SO4 4.34 g/L Proline 288 g/L Thioproline 53 mg/L Sucrose 30 g/L pH 5.8 (KOH) Phytoagar 8.0 g/L Kinetin 0.2 mg/L (add after autoclaving) 2,4D 1.5 mg/L (add after autoclaving) C. BL0 (Blaydes Media) Blaydes modified basal medium (PhytoTechnology Laboratories) L myo-inositol 100 mg/ yeast extract 2.0 g/L pH 5.8 (KOH) Phyta gel 2.5 g/L D. Half MSN medium MS macro and micro salts, half strength (Duchefa) Nitch & Nitch vitamins (Duchefa) Sucrose 15 g/L pH 5.8 (KOH) Phytoagar 8.0 g/L Transformation of Ryegrass

Ryegrass plants over-expressing the Peapod construct were generated by microprojectile bombardment using a method adapted from Altpeter et al. 2000 (Molecular Breeding 6: 519-528).

Calli for transformation were induced from immature inflorescences up to 7 mm. Floral tillers were harvested, surface sterilised in a sodium hypochlorite solution (4% available chlorine), dissected, then cultured in the dark at 25° C. for four to six weeks prior to transformation on a basal medium of Murashige and Skoog (MS) macro, micronutrients and vitamins (1962 Physiol Plant. 15: 473-497) supplemented with 30 g/L maltose, 5 mg/L 2,4-D, pH adjusted to 5.8 and solidified with 6 g/L agarose.

Plasmids were prepared using the Invitrogen Pure Link Hi Pure Plasmid Maxiprep Kit with the concentration adjusted to 1 μg/μL. The plasmid pAcH1, which contains an expression cassette comprising a chimeric hygromycin phosphotransferase (HPH) gene (Bilang et al. 1991 Gene 100: 247-250) expressed from the rice actin promoter with the first intron and terminated from the nos 3′ polyadenylation signal, was used for selection. Plasmids containing PPD expression cassettes were mixed in a 1:1 molar ratio with pAcH1.

Plasmid DNA's were coated onto M17 tungsten particles (1.4 μM diameter mean distribution) using the method of Sanford et al. 1993 (Meth. Enzymol. 217: 483-509.) and transformed into target tissues using a DuPont PDS-1000/He Biolistic Particle Delivery System. Up to 6 hours before transformation the callus was sub-cultured onto the callus initiation media containing 64 g/L mannitol. Following transformation (approximately 16 hours) transformed calli were then transferred to a mannitol-free MS basal medium supplemented with 2 mg/L 2,4-D. After 2 days calli were transferred to the same medium containing 200 mg/L hygromycin and cultured in the dark for 4 weeks for the selection of transgenic events. Regeneration of whole plants from somatic embryos occurred under lights on a MS basal medium supplemented with 0.2 mg/L Kinetin, 30 g/L, sucrose, and 50 mg/L hygromycin, adjusted to pH 5.8 and solidified with 8 g/L phytoagar. Transformed plants were transferred to a contained greenhouse environment for analysis.

PCR Analysis of Transformants

PCR analysis was performed to confirm stable integration of the HPH and PPD transgenes into the genome for plants recovered from transformation experiments. Genomic DNA was extracted from approximately 50 mg of in vitro grown leaves using the Genomic DNA Mini Kit (Geneaid). Primer pairs specific to the HPH gene (hpt-1, 5′-GCTGGGGCGTCGGTTTCCACTATCCG-3′ [SEQ ID NO: 134]; hpt-2, 5′-CGCATAACAGCGGTCATTGACTGGAGC-3′ [SEQ ID NO: 135];) and nos3′ polyadenylation signal (nos3′-1f, 5′-CTGTTGCCGGTCTTGCGATG-3′ [SEQ ID NO: 136]; nos3′-1r, 5′-GTCACATAGATGACACCGCG-3′ [SEQ ID NO: 137];) were used to produce amplification products of 375 bp and 202 bp respectively. Control reactions comprising plasmid DNA template, non-transformed plant DNA or water only were also included. The protocol for PCR reactions consisted of: an initial denaturation of 94° C. for 5 minutes, 30 cycles of 95° C. 30 s, 55° C. 15 s, 72° C. 1 min, and an extension of 72° C. for 10 min. Amplification products were resolved on 1.0% agarose gels by gel electrophoresis in TAE buffer and visualized with a Bio-Rad Gel Doc imaging system.

Southern Blot Analysis of Grass Transformants

Southern blot hybridization was used to estimate the number of transgene copies per line. Genomic DNA was extracted from leaf material of greenhouse grown plants for Southern blot hybridization using the method of Doyle J and Doyle J 1990 (Focus, 12:13-15). DNA (20 μg) was digested and separated on a 0.8% agarose gel and transferred onto a nylon membrane (Roche) using capillary transfer with 0.4N NaOH. Genomic DNAs were digested with XbaI or HindIII when probing for the HPH and PPD transgenes respectively. Probes were prepared using the DIG PCR synthesis kit. Primer pairs specific to the HPH gene (rgh1, 5′-CTCGTGCTTTCAGCTTCGATGTAG-3′ [SEQ ID NO: 138]; rgh5, 5′-GCTGGGGCGTCGGTTTCCACTATCGG-3′ [SEQ ID NO: 139]) and PPD (GrPPD1F, 5′-CACAGGATGGATTCTCCAAGG-3′ [SEQ ID NO: 140]; GrPPD1R, 5′-TAAGGTCCACGGAGAGGTTC-3′ [SEQ ID NO: 141]) were used to produce amplification products of 906 bp and 586 bp for probes respectively. Prehybridization (1 hour) and hybridization (12 hours) were performed at 45° C. using standard buffers (Roche). Detection was achieved using a non-radioactive method according to the manufacturer's protocol with CDP-Star as the chemiluminescent substrate. Light signals were detected using a Bio-Rad ChemiDoc MP System and software.

Generation of Polyclonal Antibodies Against PPD1 Protein and Immunoblotting

Custom made anti-PPD1 affinity-purified rabbit polyclonal antibodies were produced by GenScript using a full length Arabidopsis thaliana PPD1 protein. At a 1:5000 dilution the antibodies were capable of detecting less than 10 ng of purified PPD protein by immunoblot. Plant tissue was frozen in liquid nitrogen and ground to a fine powder. The frozen tissue powder was added to extraction buffer containing 50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% (vol/vol) glycerol, 5 mM DTT, 1% (vol/vol) complete protease inhibitor cocktail (Sigma), and 1% (vol/vol) Triton X-100 at a ratio of 1.0/1.5 (wt/vol), homogenised until thawed and then centrifuged for 12 min at 16,300 g and 4° C. Total soluble protein in the supernatant was quantified by Bradford assay (Coomassie Plus, Thermo Scientific), adjusted to give equivalent total protein concentrations per sample (typically between 10-40 μg), denatured in 1×NuPAGE LDS sample buffer (Invitrogen) and run in a 4-12% Bis-Tris SDS/PAGE gel (Novex). Following blotting to PVDF membrane using an iBlot apparatus (Invitrogen) protein detection was with a 1:5,000 dilution of the 1° anti-PPD1 polyclonal antibodies, followed by a 1:5,000 dilution of 2° anti-rabbit goat HRP antibodies (Sigma), application of Western Bright ECL reagent (Advansta), and image capture using a ChemiDoc™ instrument (BioRad).

Root Biomass Analysis of Transformants

An equal number (typically 4-10) of tillers were taken from WT and ryegrass plants transformed with Arabidopsis PPD under a constitutive promoter. Tillers were planted into plastic grow bags containing potting mix and pruned to be of equal height. Plants were grown for approximately 6 weeks in the glasshouse; the increase in root biomass/growth/length/branching of ryegrass plants transformed with Arabidopsis PPD under a constitutive promoter compared to WT plants could be seen by observing the root growth beyond the grow bag (FIG. 11). The increase in root biomass was quantified by washing the roots under running water then removing the attached above ground portion (leaves and stems) and drying the roots at 65° C. for 48 hr then weighing the dry weights (Table 2).

TABLE 2 Constit Constit Constit Constit promoter:: promoter:: promoter:: promoter:: wt Vector Arabidopsis Arabidopsis Arabidopsis Arabidopsis ryegrass control PPD line 1 PPD line 2 PPD line 3 PPD line 4 Av root weight (g) 0.0733 0.0387 0.4506 0.5657 0.3077 0.3503 (n = 12) SE 0.0138 0.0125 0.0428 0.0625 0.0426 0.0638

An equal number (typically 4-10) of tillers were taken from WT and ryegrass plants transformed with Arabidopsis PPD under one of three root promoters. Tillers were planted into plastic grow bags containing potting mix and pruned to be of equal height. Plants were grown for approximately 6 weeks in the glasshouse; the increase in root biomass/growth/length/branching of ryegrass plants transformed with Arabidopsis PPD under a constitutive promoter compared to WT plants could be seen by observing the root growth beyond the grow bag (FIG. 12). The increase in root biomass was quantified by washing the roots under running water then removing the attached above ground portion (leaves and stems) and drying the roots at 65° C. for 48 hr then weighing the dry weights (Table 3 and Table 4)

TABLE 3 pTobRB7Δ0.6:: pTobRB7Δ0.6:: pTobRB7Δ0.6:: Wt Vector Arabidopsis Arabidopsis Arabidopsis ryegrass control PPD line 1 PPD line 2 PPD line 3 Av root 0.0733 0.0387 0.2338 0.3686 0.3704 weight (g) (n = 12) SE 0.0138 0.0125 0.0357 0.0356 0.0611

TABLE 4 pTobRB7Δ1.3:: pTobRB7Δ1.3:: pTobRB7Δ1.3:: pTobRB7Δ1.3:: wt Vector Arabidopsis Arabidopsis Arabidopsis Arabidopsis ryegrass control PPD line 1 PPD line 2 PPD line 3 PPD line 4 Av root weight (g) 0.0733 0.0387 0.3227 0.2338 0.2720 0.4014 (n = 12) SE 0.0138 0.0125 0.0556 0.0191 0.0581 0.0445

Cuttings of approximately equal size were taken from WT and alfalfa (variety USD5) plants transformed with Arabidopsis PPD under a constitutive promoter. Cuttings were planted into plastic grow bags containing potting mix. Plants were grown for approximately 6 weeks in a growth cabinet with x hours day length; the increase in root biomass/growth/length/branching of alfalfa plants transformed with Arabidopsis PPD under a constitutive promoter compared to WT plants could be seen by observing the root growth beyond the grow bag. The increase in root biomass was quantified by washing the roots under running water (these were photographed, FIG. 12) then the attached above ground portion (leaves and stems) was removed and the roots dried at 65° C. for 48 hr then weighed Table 5.

TABLE 5 Combined dry root weights Plant line from 10 cuttings (mg) Untransformed control 43 Transformant line 1 110 Transformant line 2 54 Transformant line 3 160 Transformant line 4 59 Transformant line 5 53 Transformant line 6 85 Transformant line 7 148

An equal number (typically 4-10) of tillers were taken from WT and ryegrass plants transformed with T. repens PPD under a constitutive promoter. Tillers were planted into plastic grow bags containing potting mix and pruned to be of equal height. Plants were grown for approximately 6 weeks in the glasshouse; the increase in root biomass/growth/length/branching of ryegrass plants transformed with T. repens PPD under a constitutive promoter compared to WT plants could be seen by observing the root growth beyond the grow bag. The increase in root biomass can be quantified by washing the roots under running water then removing the attached above ground portion (leaves and stems) and drying the roots at 65° C. for 48 hr then weighing the dry weights.

An equal number (typically 4-10) of tillers were taken from WT and ryegrass plants transformed with T. repens PPD under one of three root promoters. Tillers were planted into plastic grow bags containing potting mix and pruned to be of equal height. Plants were grown for approximately 6 weeks in the glasshouse; the increase in root biomass/growth/length/branching of ryegrass plants transformed with T. repens PPD under a constitutive promoter compared to WT plants could be seen by observing the root growth beyond the grow bag. The increase in root biomass can be quantified by washing the roots under running water then removing the attached above ground portion (leaves and stems) and drying the roots at 65° C. for 48 hr then weighing the dry weights.

An equal number (typically 4-10) of tillers were taken from WT and ryegrass plants transformed with A. trichopoda PPD under a constitutive promoter. Tillers were planted into plastic grow bags containing potting mix and pruned to be of equal height. Plants were grown for approximately 6 weeks in the glasshouse; the increase in root biomass/growth/length/branching of ryegrass plants transformed with A. trichopoda PPD under a constitutive promoter compared to WT plants could be seen by observing the root growth beyond the grow bag (FIG. 11). The increase in root biomass can be quantified by washing the roots under running water then removing the attached above ground portion (leaves and stems) and drying the roots at 65° C. for 48 hr then weighing the dry weights.

An equal number (typically 4-10) of tillers were taken from WT and ryegrass plants transformed with A. trichopoda PPD under one of three root promoters. Tillers were planted into plastic grow bags containing potting mix and pruned to be of equal height. Plants were grown for approximately 6 weeks in the glasshouse; the increase in root biomass/growth/length/branching of ryegrass plants transformed with A. trichopoda PPD under a constitutive promoter compared to WT plants could be seen by observing the root growth beyond the grow bag. The increase in root biomass can be quantified by washing the roots under running water then removing the attached above ground portion (leaves and stems) and drying the roots at 65° C. for 48 hr then weighing the dry weights.

An equal number (typically 4-10) of tillers were taken from WT and ryegrass plants transformed with M. acuminate PPD under a constitutive promoter. Tillers were planted into plastic grow bags containing potting mix and pruned to be of equal height. Plants were grown for approximately 6 weeks in the glasshouse; the increase in root biomass/growth/length/branching of ryegrass plants transformed with M. acuminate PPD under a constitutive promoter compared to WT plants could be seen by observing the root growth beyond the grow bag. The increase in root biomass can be quantified by washing the roots under running water then removing the attached above ground portion (leaves and stems) and drying the roots at 65° C. for 48 hr then weighing the dry weights.

An equal number (typically 4-10) of tillers were taken from WT and ryegrass plants transformed with M. acuminate PPD under one of three root promoters. Tillers were planted into plastic grow bags containing potting mix and pruned to be of equal height. Plants were grown for approximately 6 weeks in the glasshouse; the increase in root biomass/growth/length/branching of ryegrass plants transformed with M. acuminate PPD under a constitutive promoter compared to WT plants could be seen by observing the root growth beyond the grow bag. The increase in root biomass can be quantified by washing the roots under running water then removing the attached above ground portion (leaves and stems) and drying the roots at 65° C. for 48 hr then weighing the dry weights.

An equal number (typically 4-10) of tillers were taken from WT and ryegrass plants transformed with P. sitchensis PPD under a constitutive promoter. Tillers were planted into plastic grow bags containing potting mix and pruned to be of equal height. Plants were grown for approximately 6 weeks in the glasshouse; the increase in root biomass/growth/length/branching of ryegrass plants transformed with P. sitchensis PPD under a constitutive promoter compared to WT plants could be seen by observing the root growth beyond the grow bag. The increase in root biomass can be quantified by washing the roots under running water then removing the attached above ground portion (leaves and stems) and drying the roots at 65° C. for 48 hr then weighing the dry weights.

An equal number (typically 4-10) of tillers were taken from WT and ryegrass plants transformed with P. sitchensis PPD under one of three root promoters. Tillers were planted into plastic grow bags containing potting mix and pruned to be of equal height. Plants were grown for approximately 6 weeks in the glasshouse; the increase in root biomass/growth/length/branching of ryegrass plants transformed with P. sitchensis PPD under a constitutive promoter compared to WT plants could be seen by observing the root growth beyond the grow bag. The increase in root biomass can be quantified by washing the roots under running water then removing the attached above ground portion (leaves and stems) and drying the roots at 65° C. for 48 hr then weighing the dry weights.

An equal number (typically 4-10) of tillers were taken from WT and ryegrass plants transformed with S. moellendorffii PPD under a constitutive promoter. Tillers were planted into plastic grow bags containing potting mix and pruned to be of equal height. Plants were grown for approximately 6 weeks in the glasshouse; the increase in root biomass/growth/length/branching of ryegrass plants transformed with S. moellendorffii PPD under a constitutive promoter compared to WT plants could be seen by observing the root growth beyond the grow bag. The increase in root biomass can be quantified by washing the roots under running water then removing the attached above ground portion (leaves and stems) and drying the roots at 65° C. for 48 hr then weighing the dry weights.

An equal number (typically 4-10) of tillers were taken from WT and ryegrass plants transformed with S. moellendorffii PPD under one of three root promoters. Tillers were planted into plastic grow bags containing potting mix and pruned to be of equal height. Plants were grown for approximately 6 weeks in the glasshouse; the increase in root biomass/growth/length/branching of ryegrass plants transformed with S. moellendorffii PPD under a constitutive promoter compared to WT plants could be seen by observing the root growth beyond the grow bag. The increase in root biomass can be quantified by washing the roots under running water then removing the attached above ground portion (leaves and stems) and drying the roots at 65° C. for 48 hr then weighing the dry weights.

Seeds from WT and Δppd Arabidopsis plants transformed with T. repens PPD under a constitutive promoter were scarified and sown into potting mix. Plants were grown for approximately 6 weeks in a growth cabinet with x hours day length; the increase in root biomass/growth/length/branching of Arabidopsis plants transformed with T. repens PPD under a constitutive promoter compared to WT plants could be seen by observing the root growth beyond the pot. The increase in root biomass was quantified by washing the roots under running water then the attached above ground portion (leaves and stems) was removed and the roots dried at 65° C. for 48 hr then weighed.

Seeds from WT and Δppd Arabidopsis plants transformed with M. acuminate PPD under a constitutive promoter were scarified and sown into potting mix. Plants were grown for approximately 6 weeks in a growth cabinet with x hours day length; the increase in root biomass/growth/length/branching of Arabidopsis plants transformed with M. acuminate PPD under a constitutive promoter compared to WT plants could be seen by observing the root growth beyond the pot. The increase in root biomass was quantified by washing the roots under running water then the attached above ground portion (leaves and stems) was removed and the roots dried at 65° C. for 48 hr then weighed.

Seeds from WT and Δppd Arabidopsis plants transformed with A. trichopoda PPD under a constitutive promoter were scarified and sown into potting mix. Plants were grown for approximately 6 weeks in a growth cabinet with x hours day length; the increase in root biomass/growth/length/branching of Arabidopsis plants transformed with A. trichopoda PPD under a constitutive promoter compared to WT plants could be seen by observing the root growth beyond the pot. The increase in root biomass was quantified by washing the roots under running water then the attached above ground portion (leaves and stems) was removed and the roots dried at 65° C. for 48 hr then weighed.

Seeds from WT and Δppd Arabidopsis plants transformed with P. abies PPD under a constitutive promoter were scarified and sown into potting mix. Plants were grown for approximately 6 weeks in a growth cabinet with x hours day length; the increase in root biomass/growth/length/branching of Arabidopsis plants transformed with P. abies PPD under a constitutive promoter compared to WT plants could be seen by observing the root growth beyond the pot. The increase in root biomass was quantified by washing the roots under running water then the attached above ground portion (leaves and stems) was removed and the roots dried at 65° C. for 48 hr then weighed.

Seeds from WT and Δppd Arabidopsis plants transformed with S. moellendorffii PPD under a constitutive promoter were scarified and sown into potting mix. Plants were grown for approximately 6 weeks in a growth cabinet with x hours day length; the increase in root biomass/growth/length/branching of Arabidopsis plants transformed with S. moellendorffii PPD under a constitutive promoter compared to WT plants could be seen by observing the root growth beyond the pot. The increase in root biomass was quantified by washing the roots under running water then the attached above ground portion (leaves and stems) was removed and the roots dried at 65° C. for 48 hr then weighed.

Drought Tolerance Analysis of Transformants

An equal number (typically 4-10) of tillers were taken from WT and ryegrass plants transformed with Arabidopsis PPD under a constitutive promoter. Tillers were planted in large pots containing potting mix and soil. Plants were allowed to establish in the glasshouse before half the plants of each type were subjected to water stress (typically 12% gravimetric water content, just above permanent wilting point) while the other half were kept watered (typically 22% gravimetric water content, approximately field capacity). The increased tolerance to drought stress of the PPD over expressing plants can be quantified by comparing root and shoot biomass with WT plants.

An equal number (typically 4-10) of tillers were taken from WT and ryegrass plants transformed with T. repens PPD under a constitutive promoter. Tillers were planted in large pots containing potting mix and soil. Plants were allowed to establish in the glasshouse before half the plants of each type were subjected to water stress (typically 12% gravimetric water content, just above permanent wilting point) while the other half were kept watered (typically 22% gravimetric water content, approximately field capacity). The increased tolerance to drought stress of the PPD over expressing plants can be quantified by comparing root and shoot biomass with WT plants.

An equal number (typically 4-10) of tillers were taken from WT and ryegrass plants transformed with M. acuminate PPD under a constitutive promoter. Tillers were planted in large pots containing potting mix and soil. Plants were allowed to establish in the glasshouse before half the plants of each type were subjected to water stress (typically 12% gravimetric water content, just above permanent wilting point) while the other half were kept watered (typically 22% gravimetric water content, approximately field capacity). The increased tolerance to drought stress of the PPD over expressing plants can be quantified by comparing root and shoot biomass with WT plants.

An equal number (typically 4-10) of tillers were taken from WT and ryegrass plants transformed with A. trichopoda PPD under a constitutive promoter. Tillers were planted in large pots containing potting mix and soil. Plants were allowed to establish in the glasshouse before half the plants of each type were subjected to water stress (typically 12% gravimetric water content, just above permanent wilting point) while the other half were kept watered (typically 22% gravimetric water content, approximately field capacity). The increased tolerance to drought stress of the PPD over expressing plants can be quantified by comparing root and shoot biomass with WT plants.

An equal number (typically 4-10) of tillers were taken from WT and ryegrass plants transformed with P. sitchensis PPD under a constitutive promoter. Tillers were planted in large pots containing potting mix and soil. Plants were allowed to establish in the glasshouse before half the plants of each type were subjected to water stress (typically 12% gravimetric water content, just above permanent wilting point) while the other half were kept watered (typically 22% gravimetric water content, approximately field capacity). The increased tolerance to drought stress of the PPD over expressing plants can be quantified by comparing root and shoot biomass with WT plants.

An equal number (typically 4-10) of tillers were taken from WT and ryegrass plants transformed with S. moellendorffii PPD under a constitutive promoter. Tillers were planted in large pots containing potting mix and soil. Plants were allowed to establish in the glasshouse before half the plants of each type were subjected to water stress (typically 12% gravimetric water content, just above permanent wilting point) while the other half were kept watered (typically 22% gravimetric water content, approximately field capacity). The increased tolerance to drought stress of the PPD over expressing plants can be quantified by comparing root and shoot biomass with WT plants.

An equal number (typically 4-10) of tillers were taken from WT and ryegrass plants transformed with Arabidopsis PPD under one of three root promoters. Tillers were planted in large pots containing potting mix and soil. Plants were allowed to establish in the glasshouse before half the plants of each type were subjected to water stress (typically 12% gravimetric water content, just above permanent wilting point) while the other half were kept watered (typically 22% gravimetric water content, approximately field capacity). The increased tolerance to drought stress of the PPD over expressing plants can be quantified by comparing root and shoot biomass with WT plants.

An equal number (typically 4-10) of tillers were taken from WT and ryegrass plants transformed with T. repens PPD under one of three root promoters. Tillers were planted in large pots containing potting mix and soil. Plants were allowed to establish in the glasshouse before half the plants of each type were subjected to water stress (typically 12% gravimetric water content, just above permanent wilting point) while the other half were kept watered (typically 22% gravimetric water content, approximately field capacity). The increased tolerance to drought stress of the PPD over expressing plants can be quantified by comparing root and shoot biomass with WT plants.

An equal number (typically 4-10) of tillers were taken from WT and ryegrass plants transformed with M. acuminate PPD under one of three root promoters. Tillers were planted in large pots containing potting mix and soil. Plants were allowed to establish in the glasshouse before half the plants of each type were subjected to water stress (typically 12% gravimetric water content, just above permanent wilting point) while the other half were kept watered (typically 22% gravimetric water content, approximately field capacity). The increased tolerance to drought stress of the PPD over expressing plants can be quantified by comparing root and shoot biomass with WT plants.

An equal number (typically 4-10) of tillers were taken from WT and ryegrass plants transformed with A. trichopoda PPD under one of three root promoters. Tillers were planted in large pots containing potting mix and soil. Plants were allowed to establish in the glasshouse before half the plants of each type were subjected to water stress (typically 12% gravimetric water content, just above permanent wilting point) while the other half were kept watered (typically 22% gravimetric water content, approximately field capacity). The increased tolerance to drought stress of the PPD over expressing plants can be quantified by comparing root and shoot biomass with WT plants.

An equal number (typically 4-10) of tillers were taken from WT and ryegrass plants transformed with P. sitchensis PPD under one of three root promoters. Tillers were planted in large pots containing potting mix and soil. Plants were allowed to establish in the glasshouse before half the plants of each type were subjected to water stress (typically 12% gravimetric water content, just above permanent wilting point) while the other half were kept watered (typically 22% gravimetric water content, approximately field capacity). The increased tolerance to drought stress of the PPD over expressing plants can be quantified by comparing root and shoot biomass with WT plants.

An equal number (typically 4-10) of tillers were taken from WT and ryegrass plants transformed with S. moellendorffii PPD under one of three root promoters. Tillers were planted in large pots containing potting mix and soil. Plants were allowed to establish in the glasshouse before half the plants of each type were subjected to water stress (typically 12% gravimetric water content, just above permanent wilting point) while the other half were kept watered (typically 22% gravimetric water content, approximately field capacity). The increased tolerance to drought stress of the PPD over expressing plants can be quantified by comparing root and shoot biomass with WT plants.

An equal number (typically 4-10) of WT and Δppd Arabidopsis seedlings transformed with Arabidopsis PPD under a constitutive promoter were planted in large pots containing potting mix and soil. Plants were allowed to establish in the glasshouse before half the plants of each type were subjected to water stress (typically 12% gravimetric water content, just above permanent wilting point) while the other half were kept watered (typically 22% gravimetric water content, approximately field capacity). The increased tolerance to drought stress of the PPD over expressing plants can be quantified by comparing root and shoot biomass with WT plants.

An equal number (typically 4-10) of WT and Δppd Arabidopsis seedlings transformed with T. repens PPD under a constitutive promoter were planted in large pots containing potting mix and soil. Plants were allowed to establish in the glasshouse before half the plants of each type were subjected to water stress (typically 12% gravimetric water content, just above permanent wilting point) while the other half were kept watered (typically 22% gravimetric water content, approximately field capacity). The increased tolerance to drought stress of the PPD over expressing plants can be quantified by comparing root and shoot biomass with WT plants.

An equal number (typically 4-10) of WT and Δppd Arabidopsis seedlings transformed with M. acuminate PPD under a constitutive promoter were planted in large pots containing potting mix and soil. Plants were allowed to establish in the glasshouse before half the plants of each type were subjected to water stress (typically 12% gravimetric water content, just above permanent wilting point) while the other half were kept watered (typically 22% gravimetric water content, approximately field capacity). The increased tolerance to drought stress of the PPD over expressing plants can be quantified by comparing root and shoot biomass with WT plants.

An equal number (typically 4-10) of WT and Δppd Arabidopsis seedlings transformed with A. trichopoda PPD under a constitutive promoter were planted in large pots containing potting mix and soil. Plants were allowed to establish in the glasshouse before half the plants of each type were subjected to water stress (typically 12% gravimetric water content, just above permanent wilting point) while the other half were kept watered (typically 22% gravimetric water content, approximately field capacity). The increased tolerance to drought stress of the PPD over expressing plants can be quantified by comparing root and shoot biomass with WT plants.

An equal number (typically 4-10) of WT and Δppd Arabidopsis seedlings transformed with P. abies PPD under a constitutive promoter were planted in large pots containing potting mix and soil. Plants were allowed to establish in the glasshouse before half the plants of each type were subjected to water stress (typically 12% gravimetric water content, just above permanent wilting point) while the other half were kept watered (typically 22% gravimetric water content, approximately field capacity). The increased tolerance to drought stress of the PPD over expressing plants can be quantified by comparing root and shoot biomass with WT plants.

An equal number (typically 4-10) of WT and Δppd Arabidopsis seedlings transformed with S. moellendorffii PPD under a constitutive promoter were planted in large pots containing potting mix and soil. Plants were allowed to establish in the glasshouse before half the plants of each type were subjected to water stress (typically 12% gravimetric water content, just above permanent wilting point) while the other half were kept watered (typically 22% gravimetric water content, approximately field capacity). The increased tolerance to drought stress of the PPD over expressing plants can be quantified by comparing root and shoot biomass with WT plants.

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SUMMARY OF SEQUENCES SEQ ID Sequence NO: type Species/Source Reference 1 polypeptide Arabidopsis thaliana PEAPOD 1 protein 2 polypeptide Arabidopsis thaliana PEAPOD 2 protein 3 polypeptide Populus euphratica PEAPOD protein 4 polypeptide Picea abies PEAPOD protein 5 Polypeptide Picea sitchensis PEAPOD protein 6 polypeptide Gossypium raimondii PEAPOD protein 7 polypeptide Aquilegia coerulea PEAPOD protein 1 8 polypeptide Aquilegia coerulea PEAPOD protein 2 9 polypeptide Medicago truncatula PEAPOD protein 10 polypeptide Solanum PEAPOD protein lycopersicum 11 polypeptide Trifolium repens PEAPOD protein 12 polypeptide Amborella A. Trichopoda PEAPOD protein 13 polypeptide Selaginella PEAPOD protein 1 moellendorffii 14 polypeptide Selaginella PEAPOD protein 2 moellendorffii 15 polypeptide Nicotiana tabacum PEAPOD protein 16 polypeptide Solanum tuberosum PEAPOD protein 17 polypeptide Glycine max PEAPOD protein 18 polypeptide Citrus clementine PEAPOD protein 19 polypeptide Ricinus communus PEAPOD protein 20 polypeptide Vitis vinifera PEAPOD protein 21 polypeptide Morus notabilis PEAPOD protein 22 polypeptide Phoenix dactylifera PEAPOD protein 23 polypeptide Theobroma cacao PEAPOD protein 24 polypeptide Spirodela polyrhiza PEAPOD protein 25 polypeptide Musa species PEAPOD protein 26 polypeptide Phalaenopsis PEAPOD protein aphrodite 27 polypeptide artificial internal 46 amino acid Arabidopsis PPD1 region 28 polypeptide artificial internal 46 amino acid consensus motif 1, identical residues 29 polypeptide artificial internal 46 amino acid consensus motif 2, variable residues 30 polypeptide artificial internal 27 amino acid Arabidopsis PPD1 region 31 polypeptide artificial internal 27 amino acid consensus motif 1, identical residues 32 polypeptide artificial internal 27 amino acid consensus motif 2, variable residues 33 polypeptide artificial 6 amino acid TIFY motif from Arabidopsis PPD1 34 polypeptide artificial 6 amino acid TIFY consensus motif 1, identical residues 35 polypeptide artificial 6 amino acid TIFY consensus motif 1, variable residues 36 polypeptide artificial PPD1 V5-HIS tail peptide sequence expressed in roots 37 polypeptide artificial Linker and V5-His tail peptide sequence 38 polypeptide artificial PPD1 (no tail) peptide sequence expressed in roots 39 polynucleotide Arabidopsis thaliana DGAT1 intron 3 nucleic acid sequence 40 polynucleotide artificial GenScript synthesised optimised Arabidopsis PPD1 coding region (with intron) nucleic acid sequence for expression in dicot roots under the tobacco TobRB7 Δ1.3 root promoter; the tobacco TobRB7 Δ0.6 root promoter 41 polynucleotide artificial GenScript synthesised optimised Arabidopsis PPD1 coding region (with intron) nucleic acid sequence for expression in dicot roots under Arabidopsis AtWRKY6 root promoter 42 polynucleotide artificial TobRB7 Δ1.3 promoter::attB1:: optimised Arabidopsis PPD- V5- His(INTRON)::attB2::nos terminator expression cassette nucleic acid sequence 43 polynucleotide artificial TobRB7 Δ0.6 promoter::attB1:: optimised Arabidopsis PPD- V5- His(INTRON)::attB2::nos terminator expression cassette nucleic acid sequence 44 polynucleotide artificial AtWRKY6 promoter::attB1:: optimised Arabidopsis PPD- V5- His(INTRON)::attB2::nos terminator expression cassette nucleic acid sequence 45 polynucleotide artificial TobRB7 Δ1.3 promoter::attB1:: optimised Arabidopsis PPD (INTRON)::attB2::nos terminator expression cassette 46 polynucleotide artificial TobRB7 Δ0.6 promoter::attB1:: optimised Arabidopsis PPD (INTRON)::attB2::nos terminator expression cassette 47 polynucleotide artificial AtWRKY6 promoter::attB1:: optimised Arabidopsis PPD (INTRON)::attB2::nos terminator expression cassette. 48 polynucleotide Lolium perenne DGAT1 intron 3 nucleic acid sequence 49 polynucleotide artificial GENEART synthesised rice optimised PPD1 coding region (with intron) nucleic acid sequence for expression in grass roots using the tobacco TobRB7 Δ1.3 root promoter; the tobacco TobRB7 Δ0.6 root promoter; and the Arabidopsis AtWRKY6 root promoter. 50 polynucleotide artificial TobRB7 Δ1.3 promoter::attB1::rice optimised PPD-V5- His(INTRON)::attB2::nos terminator expression cassette nucleic acid sequence 51 polynucleotide artificial TobRB7 Δ0.6 promoter::attB1::rice optimised PPD-V5- His(INTRON)::attB2::nos terminator expression cassette nucleic acid sequence 52 polynucleotide artificial AtWRKY6 promoter::attB1::rice optimised PPD-V5- His(INTRON)::attB2::nos terminator expression cassette nucleic acid sequence 53 polynucleotide artificial TobRB7 Δ1.3 promoter::attB1::rice optimised PPD (INTRON)::attB2::nos terminator expression cassette 54 polynucleotide artificial TobRB7 Δ0.6 promoter::attB1::rice optimised PPD (INTRON)::attB2::nos terminator expression cassette 55 polynucleotide artificial AtWRKY6 promoter::attB1::rice optimised PPD (INTRON)::attB2::nos terminator expression cassette 56 polypeptide artificial Yeast two Hybrid (Y2H) DNA binding domain (DBD) peptide sequence 57 polypeptide artificial Y2H activation domain (AD) peptide sequence 58 polypeptide artificial Y2H PPD1-DBD peptide sequence 59 polypeptide artificial Y2H PPD1-AD peptide sequence 60 polypeptide artificial Y2H PPD1-ppd-AD peptide sequence 61 polypeptide artificial Y2H PPD1-tify-AD peptide sequence 62 polypeptide artificial Y2H PPD1-jas*-AD peptide sequence 63 polypeptide artificial TPL peptide sequence 64 polypeptide artificial Y2H TPL-DBD peptide sequence 65 polypeptide artificial NINJA peptide sequence 66 polypeptide artificial Y2H NINJA-AD peptide sequence 67 polypeptide artificial Y2H BZR1-AD peptide sequence 68 polypeptide artificial Y2H RGA1 peptide sequence 69 polypeptide artificial Y2H RGA1-AD peptide sequence 70 polypeptide artificial Y2H PPD1-ppd-DBD peptide sequence 71 polypeptide artificial Y2H PPD1-tify-DBD peptide sequence 72 polypeptide artificial Y2H PPD1-jas*-DBD peptide sequence 73 polypeptide artificial Bimolecular Fluorescence (BiFC) nYFP peptide sequence 74 polypeptide artificial BiFC cYFP peptide sequence 75 polypeptide artificial BiFC nYFP-NINJA peptide sequence 76 polypeptide artificial BiFC nYFP-BZR1 peptide sequence 77 polypeptide artificial BiFC cYFP-PPD1 peptide sequence 78 polypeptide artificial BiFC cYFP-NINJA peptide sequence 79 polypeptide artificial BiFC cYFP-BZR1 peptide sequence 80 polypeptide artificial BiFC cYFP-PPD1-ppd peptide sequence 81 polypeptide artificial BiFC cYFP-PPD1-tify peptide sequence 82 polypeptide artificial BiFC cYFP-PPD1-jas* peptide sequence 83 polynucleotide Arabidopsis thaliana Arabidopsis thaliana PPD1 coding sequence 84 Polynucleotide Arabidopsis thaliana Arabidopsis thaliana PPD2 coding sequence 85 Polynucleotide Populus euphratica Populus trichocarpa, PPD coding sequence 86 Polynucleotide Picea abies Picea abies, PPD genomic sequence 87 Polynucleotide Gossypium raimondii Gossypium raimondii, PPD coding sequence 88 Polynucleotide Aquilegia coerulea Aquilegia coerulea, PPD coding sequence 1 89 Polynucleotide Aquilegia coerulea Aquilegia coerulea, PPD coding sequence 2 90 Polynucleotide Medicago truncatula Medicago truncatula, PPD coding sequence 91 Polynucleotide Solanum Solanum lycopersicum, lycopersicum PPD coding sequence 92 Polynucleotide Trifolium repens Trifolium repens, PPD coding sequence 93 Polynucleotide Amborella trichopoda Amborella trichopod, PPD coding sequence 94 Polynucleotide Selaginella Selaginella moellendorffii, moellendorffii PPD coding sequence 1 95 Polynucleotide Selaginella Selaginella moellendorffii, moellendorffii PPD coding sequence 2 96 Polynucleotide Nicotiana tabacum Nicotiana tabacum, PPD coding sequence 97 Polynucleotide Solanum tuberosum Solanum tuberosum, PPD coding sequence 98 Polynucleotide Glycine max Glycine max, PPD coding sequence 99 Polynucleotide Citrus clementine Citrus clementine, PPD coding sequence 100 Polynucleotide Ricinus communus Ricinus communus, PPD coding sequence 101 Polynucleotide Vitis vinifera Vitis vinifera, PPD coding sequence 102 Polynucleotide Morus notabilis Morus notabilis, PPD coding sequence 103 Polynucleotide Phoenix dactylifera Phoenix dactylifera, PPD coding sequence 104 Polynucleotide Theobroma cacao Theobroma cacao, PPD coding sequence 105 Polynucleotide Spirodela polyrhiza Spirodela polyrhiza, PPD genomic sequence 106 Polynucleotide Musa species Musa species, PPD coding sequence 107 Polynucleotide Phalaenopsis Phalaenopsis aphrodite, aphrodite PPD coding sequence 108 Polypeptide artificial Arabidopsis thaliana PPD1 + V5-His tag 109 Polypeptide artificial Trifolium repens PPD + V5-His tag 110 Polypeptide artificial Amborella trichopoda PPD + V5-His tag 111 Polypeptide artificial Musa acuminate PPD + V5-His tag 112 Polypeptide artificial Picea sitchensis PPD + V5- His tag 113 Polypeptide artificial Selaginella moellendorffii PPD + V5-His tag 114 Polypeptide artificial Arabidopsis thaliana PPD - no tag 115 Polypeptide artificial Trifolium repens PPD - no tag 116 Polypeptide artificial Amborella trichopoda PPD - no tag 117 Polypeptide artificial Musa acuminate PPD -no tag 118 Polypeptide artificial Picea abies PPD - no tag 119 Polypeptide artificial Selaginella moellendorffii PPD - no tag 120 Polynucleotide artificial Arabidopsis thaliana PPD - monocot optimised nucleic acid sequence 121 Polynucleotide artificial Trifolium repens PPD - monocot optimised nucleic acid sequence 122 Polynucleotide artificial Amborella trichopoda PPD - monocot optimised nucleic acid sequence 123 Polynucleotide artificial Musa acuminate PPD - monocot optimised nucleic acid sequence 124 Polynucleotide artificial Picea sitchensis PPD - monocot optimised nucleic acid sequence 125 Polynucleotide artificial Selaginella moellendorffii PPD - monocot optimised nucleic acid sequence 126 Polynucleotide artificial Arabidopsis thaliana PPD - dicot optimised nucleic acid sequence 127 Polynucleotide artificial Trifolium repens PPD - dicot optimised nucleic acid sequence 128 Polynucleotide artificial Amborella trichopoda PPD - dicot optimised nucleic acid sequence 129 Polynucleotide artificial Musa acuminate PPD - dicot optimised nucleic acid sequence 130 Polynucleotide artificial Picea abies PPD - dicot optimised nucleic acid sequence 131 Polynucleotide artificial Selaginella moellendorffii PPD - dicot optimised nucleic acid sequence 132 Polynucleotide artificial Primer, ocs3′-1f 133 Polynucleotide artificial Primer, ocs3′-1r 134 Polynucleotide artificial Primer, hpt-1 135 Polynucleotide artificial Primer, hpt-2 136 Polynucleotide artificial Primer, nos3′-1f 137 Polynucleotide artificial Primer, nos3′-1r 138 Polynucleotide artificial Primer, rgh1 139 Polynucleotide artificial Primer, rgh5 140 Polynucleotide artificial Primer, GrPPD1F 141 Polynucleotide artificial Primer, GrPPD1R 

The invention claimed is:
 1. A method for at least one of: a) increasing root biomass in a plant, and b) producing a plant increased root biomass, the method comprising the step of increasing the expression of at least one PEAPOD protein, or fragment thereof, in the plant.
 2. The method of claim 1 in which the increased expression is a consequence of the plant, or its ancestor plant or plant cell, being transformed with a polynucleotide encoding the PEAPOD protein, or fragment thereof.
 3. The method of claim 1 in which the plant is transgenic for at least one polynucleotide encoding and expressing the PEAPOD protein, or fragment thereof.
 4. The method of claim 1 in which the plant is transformed with at least one polynucleotide encoding a PEAPOD protein, or fragment thereof.
 5. The method of claim 1 comprising the step of transforming the plant, or transforming a plant cell which is regenerated into the plant, with a polynucleotide encoding the PEAPOD protein.
 6. The method of claim 1 which includes the additional step of testing or assessing the plant for increased root biomass.
 7. The method of claim 1 in which the PEAPOD protein, or fragment thereof, is a polypeptide comprising the sequence of at least one of SEQ ID NO: 28, 29, 31, 32, 34 and
 35. 8. The method of claim 1 in which the PEAPOD protein is a polypeptide comprising a sequence with at least 70% identity to any one of SEQ ID NO: 1 to
 26. 9. The method of claim 1 in which expression is increased by introducing a polynucleotide encoding a PEAPOD protein, or fragment thereof, into the plant cell or plant.
 10. The method of claim 9 in which the polynucleotide comprises a sequence with at least 70% identity to the coding sequence of any one of SEQ ID NO: 83-107 or a fragment thereof.
 11. The method of claim 9 in which the polynucleotide comprises a sequence with at least 70% identity to the sequence of any one of SEQ ID NO: 83-107 or a fragment thereof.
 12. The method of claim 9 in which the polynucleotide, or fragment thereof, is introduced into the plant as part of an expression construct.
 13. The method of claim 12 in which the expression construct comprises a promoter operatively linked to the polynucleotide or fragment thereof.
 14. The method of claim 13 in which the promoter is capable of driving, or drives, expression of the operatively linked polynucleotide or fragment thereof, constitutively in all tissues of the plant.
 15. The method of claim 13 in which the promoter is a tissue-preferred promoter.
 16. The method of claim 13 in which the promoter is capable of driving, or drives, expression of the operatively linked polynucleotide, or fragment thereof, in the below ground tissues of the plant.
 17. The method of claim 13 in which the promoter is a below ground tissues-preferred promoter.
 18. The method of claim 13 in which the promoter is a light-repressed promoter.
 19. The method of claim 13 in which the promoter is capable of driving, or drives, expression of the operatively linked polynucleotide, or fragment thereof, in the roots of the plant.
 20. The method of claim 13 in which the promoter is a root-preferred promoter.
 21. The method of claim 13 in which the promoter is a root-specific promoter.
 22. A plant that has increased root biomass as a result of having increased expression a PEAPOD protein, or fragment thereof, wherein expression is from a polynucleotide encoding the PEAPOD protein or fragment thereof, wherein the polynucleotide is operatively linked to a promoter that is at least one of: a) a tissue-preferred promoter, b) a below ground tissues-preferred promoter, c) a below ground tissue-specific promoter, d) a light-repressed promoter, e) a root-preferred promoter, and f) a root-specific promoter, and wherein the increased root biomass is relative to that of a control plant.
 23. The plant of claim 22 wherein expression of the PEAPOD protein, or fragment thereof, is increased as a consequence of the plant, or its ancestor plant or plant cell, having been transformed with the polynucleotide encoding the PEAPOD protein, or fragment thereof.
 24. The plant of claim 22 that is transgenic for a polynucleotide expressing the PEAPOD protein, or fragment thereof.
 25. A cell, part, propagule or progeny of the plant of claim 22 that is transgenic for the polynucleotide.
 26. A cell, part, propagule or progeny of the plant of claim 22 that is transgenic for the polynucleotide operatively linked to the promoter. 